Combination of vaccination and inhibition of the pd-1 pathway

ABSTRACT

The present invention relates to a vaccine/inhibitor combination comprising an RNA vaccine comprising at least one RNA comprising at least one open reading frame (ORF) coding for at least one antigen and a composition comprising at least one PD-1 pathway inhibitor, preferably directed against PD-1 receptor or its ligands PD-L1 and PD-L2. The present invention furthermore relates to a pharmaceutical composition and a kit of parts comprising the components of such a vaccine/inhibitor combination. Additionally the present invention relates to medical use of such a vaccine/inhibitor combination, the pharmaceutical composition and the kit of parts comprising such a vaccine/inhibitor combination, particularly for the prevention or treatment of tumor or cancer diseases or infectious diseases. Furthermore, the present invention relates to the use of an RNA vaccine in therapy in combination with a PD-1 pathway inhibitor and to the use of a PD-1 pathway inhibitor in therapy in combination with an RNA vaccine.

This application is a continuation of U.S. application Ser. No.14/769,720, filed Aug. 21, 2015, which is a national phase applicationunder 35 U.S.C. § 371 of International Application No.PCT/EP2014/000461, filed Feb. 21, 2014, which claims priority toInternational Application No. PCT/EP2013/000526, filed Feb. 22, 2013.The entire text of each of the above referenced disclosures isspecifically incorporated herein by reference.

The present invention relates to a vaccine/inhibitor combinationcomprising an RNA vaccine comprising at least one RNA comprising atleast one open reading frame (ORF) coding for at least one antigen and acomposition comprising at least one PD-1 pathway inhibitor, preferablydirected against PD-1 receptor or its ligands PD-L1 and/or PD-L2. Thepresent invention furthermore relates to a pharmaceutical compositionand a kit of parts comprising such a vaccine/inhibitor combination.Additionally, the present invention relates to the medical use of such avaccine/inhibitor combination, the pharmaceutical composition and thekit of parts comprising such a vaccine/inhibitor combination,particularly for the prevention or treatment of tumor or cancer diseasesor infectious diseases. Furthermore, the present invention relates tothe use of an RNA vaccine in therapy in combination with a PD-1 pathwayinhibitor and to the use of a PD-1 pathway inhibitor in therapy incombination with an RNA vaccine.

Traditionally, cancer immunotherapy was focused on stimulating theimmune system through vaccination or adoptive cellular immunotherapy toelicit an anti-tumor response. This approach was based on the assumptionthat tumor cells express antigenic targets but that anti-tumor T cellswere not sufficiently activated. Therefore, to circumvent this problem,it was mainly tried to increase the recognition of these antigenictargets by stimulating key positive co-stimulatory and innate immunepathways (such as CD28, CD154 and TLR receptors).

More recently, it became clear that the immune system does recognizetumor antigens but remains quiescent although tumor antigens arepresent. This observation led to the hypothesis that there are specificinhibitory mechanisms which limit or even shut down the anti-tumorresponse. This hypothesis was confirmed when negative regulatory T cellsurface molecules were discovered which are upregulated in activated Tcells to dampen their activity, resulting in less effective killing oftumor cells. These inhibitory molecules were termed negativeco-stimulatory molecules due to their homology to the T cellco-stimulatory molecule CD28. These proteins, also referred to as immunecheckpoint proteins, function in multiple pathways including theattenuation of early activation signals, competition for positiveco-stimulation and direct inhibition of antigen presenting cells(Bour-Jordan et al., 2011. Immunol Rev. 241(1):180-205; PMID: 21488898).One member of this protein family is programmed death-1 (PD-1) and itsligands B7-H1/PD-L1 (CD274) and B7-DC/PD-L2 (CD273).

PD-1 is expressed on activated T and B cells as well as on monocytesinvolved in regulating the balance between immune activation andtolerance. The major role of PD-1 is to limit the activity of T cells inthe periphery during an inflammatory response to infection and to limitautoimmunity. The basis for this regulation is that the PD-1 ligandsB7-H1/PD-L1 and B7-DC/PD-L2 are upregulated in response to variousproinflammatory cytokines and can bind to PD-1 on activated T cells ininflamed tissues, thereby limiting the immune response. Deletion of thePD-1 gene leads to autoimmune complications, for example lupus-likesymptoms (Nishimura et al., 1999. Immunity 11(2): 141-51; PMID:10485649).

In addition, it was found that B7-H1/PD-L1 is frequently upregulated onmany different tumor types, where it inhibits local anti-tumor T cellresponses by binding to PD-1 on tumor infiltrating lymphocytes. Forexample, it was demonstrated that PD-L1 plays a role in tumor immuneevasion in squamous cell carcinomas of the head and neck (Dong et al.,2002. Nat Med. 8(8):793-800). The authors showed that forced expressionof B7-H1/PD-L1 in tumors that are normally B7-H1/PD-L1 negative inhibitstheir immune elimination and that antibody blockade restores anti-tumorresponses. Furthermore, it was shown that many human tumors, such ascarcinomas of the lung, ovary and colon, upregulate B7-H1/PD-L1 relativeto their normal tissue counterparts. In summary, these observationssuggested to use blockade of the PD-1 pathway for cancer immunotherapy(Zitvogel and Kroemer, 2012. Oncoimmunology 1, 1223-1125).

In this context US2010/0055102 describes the use of PD-1 antagonists forincreasing a T cell immune response in a mammal.

Recently, the first clinical trials with immune checkpoint inhibitors(ICIs) against the PD-1 receptor and its ligand PD-L1 have beenreported. The first phase I trial with the anti-PD-1 monoclonal antibodyBMS-936558 was conducted in patients with treatment-refractorymetastatic solid tumors. Clinical activity was observed in patients withmelanoma, renal carcinoma, colorectal cancer and non-small cell lungcancer (NSCLC). It could be shown that PD-L1 is overexpressed in manycancers and is often associated with poor prognosis. Furthermore tumorcell surface expression of PD-L1 in pretreatment biopsies emerged as apotential biomarker of response, consistent with pathway biology(Brahmer et al., 2010. J Clin Oncol. 28(19):3167-75; PMID: 20516446).

Another clinical study with BMS-936558 produced objective responses inapproximately one in four to one in five patients with non-small-celllung cancer, melanoma, or renal-cell cancer; the adverse-event profiledoes not appear to preclude its use. Preliminary data suggest arelationship between PD-L1 expression on tumor cells and objectiveresponse (Topalian et al., 2012. N Engl J Med. 366(26):2443-54; PMID:22658127).

Furthermore, a phase I study with the anti-PD-L1 antibody BMS-936559induced durable tumor regression (objective response rate of 6 to 17%)and prolonged stabilization of disease (rates of 12 to 41% at 24 weeks)in patients with advanced cancers, including non-small-cell lung cancer,melanoma, and renal-cell cancer. Specifically, an objective response (acomplete or partial response) was observed in 9 of 52 patients withmelanoma, 2 of 17 with renal-cell cancer, 5 of 49 with non-small-celllung cancer, and 1 of 17 with ovarian cancer (Brahmer et al. 2012. NEngl J Med. 366(26):2455-65; PMID: 22658128).

Nevertheless, only relatively low responses of some cancers could bedemonstrated in these studies (e.g. lung and prostate cancer) to PD-1pathway checkpoint inhibitors. In more recent studies combinations withPD-1 pathway checkpoint inhibitors have been evaluated in preclinicalmodels.

A study reported e.g. the generation of a dendritic cell (DC) vaccinewith a combination of a PD-1 ligand siRNA and target antigen mRNA. ThesePD-L1-silenced DC's loaded with antigen mRNA increased ex vivoantigen-specific CD8+ T cell responses from transplanted cancer patients(Hobo et al. 2013. Cancer Immunol Immunother. 62(2):285-97; PMID:22903385)

Likewise, Dai et al. showed that a dendritic cell-directed lentiviralvector (DCLV) system encoding the human immunodeficiency virus (HIV)-1Gag protein combined with blocking of the PD-1/PD-1 ligand (PD-L)inhibitory signal via an anti-PD-L1 antibody generated an enhanced HIV-1Gag-specific CD8+ immune response following DCLV immunization (Dai etal. 2012. Mol Ther. 20(9):1800-9; PMID: 22588271).

Another study examined the combination of recombinant lentivector (rLV)vaccination with PD-1 and PD-L1 blocking antibodies. Combining blockingantibodies with rLV vaccination delayed tumor growth but was notsufficient to induce the complete rejection of established tumors(Sierro et al. 2011. Eur J Immunol.;41(8):2217-28; PMID: 21538347).

Additionally, WO2008/11344 discloses the modulation of an immuneresponse by combination of a DNA-based recombinant adenoviral construct(rAAV) coding for an antigen or therapeutic protein and a nucleic acidcoding for a modulator of PD-1 signaling, particulary siRNA, antisenseRNA or a ribozyme specific for PD-L1 gene.

In this context also the fusion of soluble PD-1 protein and antigenicprotein fragments was studied (WO2012/062218).

A further preclinical study reported that combination blockade of theCTLA-4- and PD-1-negative costimulatory receptors leads to synergisticlevels of tumor rejection in the context of a suitable vaccine (Curranet al., 2010. Proc Natl Acad Sci USA 107(9):4275-80; PMID: 20160101).Mice preimplanted with B16 melanoma cells were vaccinated withirradiated B16 melanoma cells expressing either GM-CSF (Gvax) orFlt3-ligand (Fvax) combined with antibody blockade of the negativeT-cell costimulatory receptor cytotoxic T-lymphocyte antigen-4 (CTLA-4),programmed death-1 (PD-1) and its ligand PD-L 1. In the context of Gvax,neither blockade of any single co-inhibitory receptor nor of anycombination resulted in greater than 20% survival. Using Fvax, blockadeof either PD-L1 (8% survival), CTLA-4 (10% survival), or PD-1 (25%survival) showed modest therapeutic benefit. However, only combinedblockade of CTLA-4 and PD-1 led to tumor rejection in 50% of mice andfurther addition of anti-PD-L1 antibody increased the rate to 65%. Insummary, only CTLA-4-blockade combined with PD-1 blockade had asignificant effect in promoting the rejection of B16 melanomas inconjunction with the B16 melanoma cell vaccine. Blocking of a singleinhibitory receptor (PD-1 or CTLA-4) leads to upregulation of theunblocked pathway. Therefore, this study suggests that a simultaneousblockade of multiple negative co-stimulatory receptors may be requiredto enable tumor-infiltrating T cells to be effective. Furthermore, basedon their preclinical data in the B16 melanoma model, the authors suggestthat combining these agents may have synergistic effects in drivingtumor rejection but they remark that combining multiple co-inhibitoryblocking antibodies in the clinic should proceed with great caution.

Mkrtichya et al. additionally reported synergistic antigen-specificimmune responses by using an anti-PD-1 antibody (CT-011) with Treg-celldepletion by low-dose cyclophosphamide (CPM), combined with a tumorvaccine (HPV16-E7 peptide vaccine). But only the combination of these 3treatments resulted in a complete regression of tumors (Mkrtichya et al.2011. Eur J Immunol. 41(10):2977-86; PMID: 21710477)

A further report examining the effect of PD-L1 inhibition on tumorgrowth showed that the systemic administration of anti-PD-L1 antibodyplus melanoma peptide-pulsed dendritic cells (DCs) resulted in a highernumber of melanoma peptide-specific CD8+ T cells, but that thiscombination was insufficient to delay the growth of established B16melanoma. Although the additional body irradiation delayed tumor growth,further adoptive transfer of antigen-specific CD8+ T cells was needed toachieve tumor regression and long-term survival of the treated mice(Pilon-Thomas et al. 2010. J Immunol. 1; 184(7):3442-9; PMID: 20194714).

In a preclinical study Fotin-Mleczek et al. showed the beneficialcombination of a tumour vaccine based on mRNA and an antibody directedagainst the CTLA4 receptor which attenuates the signaling of T cells(Fotin-Mleczek et al., 2012. J Gene Med. 14(6):428-39; PMID: 22262664).

In summary, the use of immune checkpoint inhibitors appears to representa promising approach for improved cancer immunotherapy. However, thecombination of vaccines with a single ICI often did not lead to thehoped improvement of the immunotherapy and the combined clinical use ofICIs targeting multiple negative co-stimulatory receptors or thecombination of ICIs with other treatments may induce clinicalcomplications as e.g. induction of an autoimmune disease.

Therefore, it is the object of the present invention to provide safe andeffective means for a therapy based on ICIs, particularly based on PD-1pathway inhibitors, in particular for a therapy of tumor, cancer and/orinfectious diseases.

The object underlying the present invention is solved by the claimedsubject matter. In particular, the object of the invention is solved bythe provision of a vaccine/inhibitor combination comprising as vaccinean RNA vaccine comprising at least one RNA comprising at least one openreading frame coding for at least one antigen and as inhibitor a PD-1pathway inhibitor, preferably directed against PD-1 receptor or itsligands PD-L1 and/or PD-L2. Furthermore, the object is solved by apharmaceutical composition or a kit of parts comprising thevaccine/inhibitor combination or the respective components thereof.Additionally, the object is solved by a combination of an RNA vaccinewith ICIs, particularly PD-1 pathway inhibitors for use in a method oftreatment of tumour or cancer diseases or infection diseases.

For the sake of clarity and readability the following definitions areprovided. Any technical feature mentioned for these definitions may beread on each and every embodiment of the invention. Additionaldefinitions and explanations may be specifically provided in the contextof these embodiments.

Immune response: An immune response may typically either be a specificreaction of the adaptive immune system to a particular antigen (socalled specific or adaptive immune response) or an unspecific reactionof the innate immune system (so called unspecific or innate immuneresponse). In essence, the invention is associated with specificreactions (adaptive immune responses) of the adaptive immune system.However, this specific response can be supported by an additionalunspecific reaction (innate immune response). Therefore, the inventionalso relates to a compound, composition or combination for simultaneousstimulation of the innate and the adaptive immune system to evoke anefficient adaptive immune response.

Immune system: The immune system may protect organisms from infection.If a pathogen succeeds in passing a physical barrier of an organism andenters this organism, the innate immune system provides an immediate,but non-specific response. If pathogens evade this innate response,vertebrates possess a second layer of protection, the adaptive immunesystem. Here, the immune system adapts its response during an infectionto improve its recognition of the pathogen. This improved response isthen retained after the pathogen has been eliminated, in the form of animmunological memory, and allows the adaptive immune system to mountfaster and stronger attacks each time this pathogen is encountered.According to this, the immune system comprises the innate and theadaptive immune system. Each of these two parts typically contains socalled humoral and cellular components.

Adaptive immune response: The adaptive immune response is typicallyunderstood to be an antigen-specific response of the immune system.Antigen specificity allows for the generation of responses that aretailored to specific pathogens or pathogen-infected cells. The abilityto mount these tailored responses is usually maintained in the body by“memory cells”. Should a pathogen infect the body more than once, thesespecific memory cells are used to quickly eliminate it. In this context,the first step of an adaptive immune response is the activation of naiveantigen-specific T cells or different immune cells able to induce anantigen-specific immune response by antigen-presenting cells. Thisoccurs in the lymphoid tissues and organs through which naive T cellsare constantly passing. The three cell types that may serve asantigen-presenting cells are dendritic cells, macrophages, and B cells.Each of these cells has a distinct function in eliciting immuneresponses. Dendritic cells may take up antigens by phagocytosis andmacropinocytosis and may become stimulated by contact with e.g. aforeign antigen to migrate to the local lymphoid tissue, where theydifferentiate into mature dendritic cells. Macrophages ingestparticulate antigens such as bacteria and are induced by infectiousagents or other appropriate stimuli to express MHC molecules. The uniqueability of B cells to bind and internalize soluble protein antigens viatheir receptors may also be important to induce T cells. MHC-moleculesare, typically, responsible for presentation of an antigen to T-cells.Therein, presenting the antigen on MHC molecules leads to activation ofT cells which induces their proliferation and differentiation into armedeffector T cells. The most important function of effector T cells is thekilling of infected cells by CD8+ cytotoxic T cells and the activationof macrophages by Th1 cells which together make up cell-mediatedimmunity, and the activation of B cells by both Th2 and Th1 cells toproduce different classes of antibody, thus driving the humoral immuneresponse. T cells recognize an antigen by their T cell receptors whichdo not recognize and bind the antigen directly, but instead recognizeshort peptide fragments e.g. of pathogen-derived protein antigens, e.g.so-called epitopes, which are bound to MHC molecules on the surfaces ofother cells.

Adaptive immune system: The adaptive immune system is essentiallydedicated to eliminate or prevent pathogenic growth. It typicallyregulates the adaptive immune response by providing the vertebrateimmune system with the ability to recognize and remember specificpathogens (to generate immunity), and to mount stronger attacks eachtime the pathogen is encountered. The system is highly adaptable becauseof somatic hypermutation (a process of accelerated somatic mutations),and V(D)J recombination (an irreversible genetic recombination ofantigen receptor gene segments). This mechanism allows a small number ofgenes to generate a vast number of different antigen receptors, whichare then uniquely expressed on each individual lymphocyte. Because thegene rearrangement leads to an irreversible change in the DNA of eachcell, all of the progeny (offspring) of such a cell will then inheritgenes encoding the same receptor specificity, including the Memory Bcells and Memory T cells that are the keys to long-lived specificimmunity.

Cellular immunity/cellular immune response: Cellular immunity relatestypically to the activation of macrophages, natural killer cells (NK),antigen-specific cytotoxic T-lymphocytes, and the release of variouscytokines in response to an antigen. In more general terms, cellularimmunity is not based on antibodies, but on the activation of cells ofthe immune system. Typically, a cellular immune response may becharacterized e.g. by activating antigen-specific cytotoxicT-lymphocytes that are able to induce apoptosis in cells, e.g. specificimmune cells like dendritic cells or other cells, displaying epitopes offoreign antigens on their surface. Such cells may be virus-infected orinfected with intracellular bacteria, or cancer cells displaying tumorantigens. Further characteristics may be activation of macrophages andnatural killer cells, enabling them to destroy pathogens and stimulationof cells to secrete a variety of cytokines that influence the functionof other cells involved in adaptive immune responses and innate immuneresponses.

Humoral immunity/humoral immune response: Humoral immunity referstypically to antibody production and optionally to accessory processesaccompanying antibody production. An humoral immune response may betypically characterized, e.g., by Th2 activation and cytokineproduction, germinal center formation and isotype switching, affinitymaturation and memory cell generation. Humoral immunity also typicallymay refer to the effector functions of antibodies, which includepathogen and toxin neutralization, classical complement activation, andopsonin promotion of phagocytosis and pathogen elimination.

Innate immune system: The innate immune system, also known asnon-specific (or unspecific) immune system, typically comprises thecells and mechanisms that defend the host from infection by otherorganisms in a non-specific manner. This means that the cells of theinnate system may recognize and respond to pathogens in a generic way,but unlike the adaptive immune system, it does not confer long-lastingor protective immunity to the host. The innate immune system may be,e.g., activated by ligands of Toll-like receptors (TLRs) or otherauxiliary substances such as lipopolysaccharides, TNF-alpha, CD40ligand, or cytokines, monokines, lymphokines, interleukins orchemokines, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10,IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20,IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30,IL-31, IL-32, IL-33, IFN-alpha, IFN-beta, IFN-gamma, GM-CSF, G-CSF,M-CSF, LT-beta, TNF-alpha, growth factors, and hGH, a ligand of humanToll-like receptor TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9,TLR10, a ligand of murine Toll-like receptor TLR1, TLR2, TLR3, TLR4,TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12 or TLR13, a ligand ofa NOD-like receptor, a ligand of a RIG-I like receptor, animmunostimulatory nucleic acid, an immunostimulatory RNA (isRNA), aCpG-DNA, an antibacterial agent, or an anti-viral agent. Thevaccine/inhibitor combination, the pharmaceutical composition or the kitof parts according to the present invention may comprise one or moresuch substances. Typically, a response of the innate immune systemincludes recruiting immune cells to sites of infection, through theproduction of chemical factors, including specialized chemicalmediators, called cytokines; activation of the complement cascade;identification and removal of foreign substances present in organs,tissues, the blood and lymph, by specialized white blood cells;activation of the adaptive immune system; and/or acting as a physicaland chemical barrier to infectious agents.

Adjuvant/adjuvant component: An adjuvant or an adjuvant component in thebroadest sense is typically a pharmacological and/or immunological agentthat may modify, e.g. enhance, the effect of other agents, such as adrug or vaccine. It is to be interpreted in a broad sense and refers toa broad spectrum of substances. Typically, these substances are able toincrease the immunogenicity of antigens. For example, adjuvants may berecognized by the innate immune systems and, e.g., may elicit an innateimmune response. “Adjuvants” typically do not elicit an adaptive immuneresponse. Insofar, “adjuvants” do not qualify as antigens. Their mode ofaction is distinct from the effects triggered by antigens resulting inan adaptive immune response.

Antigen: In the context of the present invention “antigen” referstypically to a substance which may be recognized by the immune system,preferably by the adaptive immune system, and is capable of triggeringan antigen-specific immune response, e.g. by formation of antibodiesand/or antigen-specific T cells as part of an adaptive immune response.Typically, an antigen may be or may comprise a peptide or protein whichcomprises at least one epitope and which may be presented by the MHC toT cells. In the sense of the present invention an antigen may be theproduct of translation of a provided RNA, preferably an mRNA as definedherein. In this context, also fragments, variants and derivatives ofpeptides and proteins comprising at least one epitope are understood asantigens. In the context of the present invention, tumour antigens andpathogenic antigens as defined herein are particularly preferred.

Epitope: Epitopes (also called ‘antigen determinant’) can bedistinguished in T cell epitopes and B cell epitopes. T cell epitopes orparts of the proteins in the context of the present invention maycomprise fragments preferably having a length of about 6 to about 20 oreven more amino acids, e.g. fragments as processed and presented by MHCclass I molecules, preferably having a length of about 8 to about 10amino acids, e.g. 8, 9, or 10, (or even 11, or 12 amino acids), orfragments as processed and presented by MHC class II molecules,preferably having a length of about 13 or more amino acids, e.g. 13, 14,15, 16, 17, 18, 19, 20 or even more amino acids, wherein these fragmentsmay be selected from any part of the amino acid sequence. Thesefragments are typically recognized by T cells in form of a complexconsisting of the peptide fragment and an MHC molecule, i.e. thefragments are typically not recognized in their native form. B cellepitopes are typically fragments located on the outer surface of(native) protein or peptide antigens as defined herein, preferablyhaving 5 to 15 amino acids, more preferably having 5 to 12 amino acids,even more preferably having 6 to 9 amino acids, which may be recognizedby antibodies, i.e. in their native form.

Such epitopes of proteins or peptides may furthermore be selected fromany of the herein mentioned variants of such proteins or peptides. Inthis context antigenic determinants can be conformational ordiscontinuous epitopes which are composed of segments of the proteins orpeptides as defined herein that are discontinuous in the amino acidsequence of the proteins or peptides as defined herein but are broughttogether in the three-dimensional structure or continuous or linearepitopes which are composed of a single polypeptide chain.

Vaccine: A vaccine is typically understood to be a prophylactic ortherapeutic material providing at least one antigen, preferably animmunogen. “Providing at least on antigen” means, for example, that thevaccine comprises the antigen or that the vaccine comprises a moleculethat, e.g., codes for the antigen or a molecule comprising the antigen.For example, the vaccine may comprise a nucleic acid, such as an RNA(e.g. RNA vaccine), which codes for a peptide or protein that comprisesthe antigen. The antigen or immunogen may be derived from any materialthat is suitable for vaccination. For example, the antigen or immunogenmay be derived from a pathogen, such as from bacteria or virus particlesetc., or from a tumor or cancerous tissue. The antigen or immunogenstimulates the body's adaptive immune system to provide an adaptiveimmune response. In the context, of the present invention, the vaccinepreferably does not comprise cells, such as dendritic cells or cancercells, e.g., B16 melanoma cells. Furthermore, in the context of thepresent invention the vaccine preferably does not consist of peptideantigens, such as peptide tumor antigens. In the context of the presentinvention, the vaccine is preferably an RNA vaccine.

RNA vaccine: An RNA vaccine is defined herein as a vaccine comprising atleast one RNA molecule comprising at least one open reading frame (ORF)coding for at least one antigen. In the context of the presentinvention, the at least one RNA molecule comprised by the vaccine ispreferably an isolated RNA molecule. This at least one RNA is preferablyviral RNA, self-replicating RNA (replicon) or most preferably mRNA. Alsoincluded herein are RNA/DNA hybrids which means that the at least oneRNA molecule of the RNA vaccine consists partially of ribonucleotidesand partially of deoxyribonucleotides. In this context, the at least oneRNA of the RNA vaccine consists to at least 50% of ribonucleotides, morepreferably to at least 60%, 70%, 80%, 90% and most preferably to 100%.In this context, the at least one RNA of the RNA vaccine can also beprovided as complexed RNA or mRNA, as virus particle and as repliconparticle as defined herein. In the context of the present invention, theat least one RNA comprised by the RNA vaccine is preferably not presentin cells, such as in dendritic cell or cancer cells, e.g. B16 melanomacells. Furthermore it is particularly preferred that the RNA vaccine ofthe invention does not comprise or does not consist of a lentiviralvector, in particular not a dendritic cell-directed lentiviral vector,or an (recombinant) adenoviral/adeno-associated vector ((r)AAV vector).Preferably, the RNA vaccine of the invention is not an HIV vaccine. Inparticular, it is preferred that the RNA vaccine of the invention doesnot comprise an AAV or lentiviral vector encoding one or more antigens.Also, the RNA vaccine of the invention may preferably not encodeHIV-specific antigens, e.g. Gag protein, in particular not a lentiviralvector encoding HIV specific antigens, like Gag. Preferably, the RNAvaccine of the invention does not comprise a fusion protein of a PD1protein and an antigenic protein or a fusion protein of a PD1 proteinand an immunoglobulin or a portion thereof and does not encode such aPD1 fusion protein.

Genetic vaccination: Genetic vaccination may typically be understood tobe vaccination by administration of a nucleic acid molecule encoding anantigen or an immunogen or fragments thereof. The nucleic acid moleculemay be administered to a subject's body or to isolated cells of asubject. Upon transfection of certain cells of the body or upontransfection of the isolated cells, the antigen or immunogen may beexpressed by those cells and subsequently presented to the immunesystem, eliciting an adaptive, i.e. antigen-specific immune response.Accordingly, genetic vaccination typically comprises at least one of thesteps of a) administration of a nucleic acid, preferably an (isolated)RNA as defined herein, to a subject, preferably a patient, or toisolated cells of a subject, preferably a patient, which usually resultsin transfection of the subject's cells either in vivo or in vitro; b)transcription and/or translation of the introduced nucleic acidmolecule; and optionally c) re-administration of isolated, transfectedcells to the subject, preferably the patient, if the nucleic acid hasnot been administered directly to the patient.

Nucleic acid: The term nucleic acid means any DNA- or RNA-molecule andis used synonymous with polynucleotide. Furthermore, modifications orderivatives of the nucleic acid as defined herein are explicitlyincluded in the general term “nucleic acid”. For example, PNA is alsoincluded in the term “nucleic acid”.

Monocistronic RNA: A monocistronic RNA may typically be an RNA,preferably an mRNA, that comprises only one open reading frame. An openreading frame in this context is a sequence of several nucleotidetriplets (codons) that can be translated into a peptide or protein.

Bi-/multicistronic RNA: RNA, preferably mRNA, that typically may havetwo (bicistronic) or more (multicistronic) open reading frames (ORF). Anopen reading frame in this context is a sequence of several nucleotidetriplets (codons) that can be translated into a peptide or protein.

5′-Cap structure: A 5′ Cap is typically a modified nucleotide,particularly a guanine nucleotide, added to the 5′ end of an RNAmolecule. Preferably, the 5′-Cap is added using a 5′-5′-triphosphatelinkage.

Poly(C) sequence: A poly(C) sequence is typically a long sequence ofcytosine nucleotides, typically about 10 to about 200 cytidinenucleotides, preferably about 10 to about 100 cytidine nucleotides, morepreferably about 10 to about 70 cytidine nucleotides or even morepreferably about 20 to about 50 or even about 20 to about 30 cytidinenucleotides. A poly(C) sequence may preferably be located 3′ of thecoding region comprised by a nucleic acid.

Poly(A) tail: A poly(A) tail also called “3′-poly(A) tail” is typicallya long sequence of adenine nucleotides of up to about 400 adenosinenucleotides, e.g. from about 25 to about 400, preferably from about 50to about 400, more preferably from about 50 to about 300, even morepreferably from about 50 to about 250, most preferably from about 60 toabout 250 adenosine nucleotides, added to the 3′ end of a nucleic acidsequence, preferably an mRNA. A poly(A) tail may preferably be located3′ of the coding region comprised by a nucleic acid, e.g. an mRNA.

Stabilized nucleic acid: A stabilized nucleic acid, typically, may beessentially resistant to in vivo degradation (e.g. degradation by anexo- or endo-nuclease) and/or ex vivo degradation (e.g. by themanufacturing process prior to vaccine administration, e.g. in thecourse of the preparation of the RNA vaccine solution to beadministered). Stabilization of RNA, particularly mRNA can, e.g., beachieved by providing a 5′-Cap structure, a Poly(A) tail, a poly (C)tail, and/or any other UTR modification. It can also be achieved bybackbone modification, sugar modification, base modification, and/ormodification of the G/C-content of the nucleic acid. Various othermethods are conceivable in the context of the invention. Modification ofa nucleic acid (modified nucleic acid): Modification of a nucleic acidmolecule, particularly of RNA or mRNA, may contain backbonemodifications, sugar modifications or base modifications. A backbonemodification in connection with the present invention is a modificationin which phosphates of the backbone of the nucleotides contained in thenucleic acid molecule are chemically modified. A sugar modification inconnection with the present invention is a chemical modification of thesugar of the nucleotides of the nucleic acid. Furthermore, a basemodification in connection with the present invention is a chemicalmodification of the base moiety of the nucleotides of the nucleic acidmolecule. Therefore a modified nucleic acid is also defined herein as anucleic acid molecule which may include nucleotide analogues.Furthermore a modification of a nucleic acid molecule can contain alipid modification. Such a lipid-modified nucleic acid typicallycomprises a nucleic acid as defined herein. Such a lipid-modifiednucleic acid molecule typically further comprises at least one linkercovalently linked with that nucleic acid molecule, and at least onelipid covalently linked with the respective linker. Alternatively, thelipid-modified nucleic acid molecule comprises at least one nucleic acidmolecule as defined herein and at least one (bifunctional) lipidcovalently linked (without a linker) with that nucleic acid molecule.According to a third alternative, the lipid-modified nucleic acidmolecule comprises a nucleic acid molecule as defined herein, at leastone linker covalently linked with that nucleic acid molecule, and atleast one lipid covalently linked with the respective linker, and alsoat least one (bifunctional) lipid covalently linked (without a linker)with that nucleic acid molecule. A modification of a nucleic acid mayalso comprise the modification of the G/C content of the coding regionof a nucleic acid molecule, especially of the at least one RNA of theRNA vaccine encoding at least one antigen in the inventivevaccine/inhibitor combination. In this context it is particularlypreferred that the G/C content of the coding region of the nucleic acidmolecule is increased, compared to the G/C content of the coding regionof its particular wild type coding sequence, i.e. the unmodified RNA.The encoded amino acid sequence of the nucleic acid sequence ispreferably not modified compared to the coded amino acid sequence of theparticular wild type mRNA. The modification of the G/C-content of thenucleic acid molecule, especially if the nucleic acid molecule is in theform of an mRNA or codes for an mRNA, is based on the fact that thesequence of any mRNA region to be translated is important for efficienttranslation of that mRNA. Thus, the composition and the sequence ofvarious nucleotides are important. In particular, sequences having anincreased G (guanosine)/C (cytosine) content are more stable thansequences having an increased A (adenosine)/U (uracil) content.Therefore, the codons of the coding sequence or mRNA are thereforevaried compared to its wild type coding sequence or mRNA, whileretaining the translated amino acid sequence, such that they include anincreased amount of G/C nucleotides. In respect to the fact that severalcodons code for one and the same amino acid (so-called degeneration ofthe genetic code), the most favourable codons for the stability can bedetermined (so-called alternative codon usage).Preferably, the G/Ccontent of the coding region of the nucleic acid molecule, especially ofthe at least one RNA of the RNA vaccine encoding at least one antigen inthe inventive vaccine/inhibitor combination, is increased by at least7%, more preferably by at least 15%, particularly preferably by at least20%, compared to the G/C content of the coded region of the wild typemRNA. According to a specific embodiment at least 5%, 10%, 20%, 30%,40%, 50%, 60%, more preferably at least 70%, even more preferably atleast 80% and most preferably at least 90%, 95% or even 100% of thesubstitutable codons in the region coding for a protein or peptide asdefined herein or its fragment, variant and/or derivative thereof or thewhole sequence of the wild type mRNA sequence or coding sequence aresubstituted, thereby increasing the G/C content of said sequence. Inthis context, it is particularly preferable to increase the G/C contentof the nucleic acid molecule, especially of the at least one RNA of theRNA vaccine encoding at least one antigen in the inventivevaccine/inhibitor combination, to the maximum (i.e. 100% of thesubstitutable codons), in particular in the region coding for a protein,compared to the wild type sequence. Furthermore a modification of thenucleic acid, especially of the at least one RNA of the RNA vaccineencoding at least one antigen in the inventive vaccine/inhibitorcombination, is based on the finding that the translation efficiency isalso determined by a different frequency in the occurrence of tRNAs incells. The frequency in the occurrence of tRNAs in a cell, and thus thecodon usage in said cell, is dependent on the species the cell isderived from. Accordingly, a yeast cell generally exhibits a differentcodon usage than a mammalian cell, such as a human cell. Thus, ifso-called “rare codons” are present in the nucleic acid molecule (withrespect to the respective expression system), especially if the nucleicacid is in the form of an mRNA or codes for an mRNA, to an increasedextent, the corresponding modified nucleic acid molecule is translatedto a significantly poorer degree than in the case where codons codingfor relatively “frequent” tRNAs are present. Therefore, the codingregion of the modified nucleic acid, particularly the at least one RNAof the RNA vaccine encoding at least one antigen in the inventivevaccine/inhibitor combination is preferably modified compared to thecorresponding region of the wild type mRNA or coding sequence such thatat least one codon of the wild type sequence which codes for a tRNAwhich is relatively rare in the cell is exchanged for a codon whichcodes for a tRNA which is relatively frequent in the cell and carriesthe same amino acid as the relatively rare tRNA. By this modification,the sequences of the nucleic acid molecule, particularly of the at leastone RNA of the RNA vaccine encoding at least one antigen in theinventive vaccine/inhibitor combination, is modified such that codonsfor which frequently occurring tRNAs are available are inserted. Inother words, by this modification all codons of the wild type sequencewhich code for a tRNA which is relatively rare in the cell can in eachcase be exchanged for a codon which codes for a tRNA which is relativelyfrequent in the cell and which, in each case, carries the same aminoacid as the relatively rare tRNA. Such a modified nucleic acid,preferably is termed herein as “codon-optimized nucleic acid or RNA”.Which tRNAs occur relatively frequently in the cell and which, incontrast, occur relatively rarely is known to a person skilled in theart; cf. e.g. Akashi, Curr. Opin. Genet. Dev. 2001, 11(6): 660-666. Itis particularly preferred that a nucleic acid sequence coding for aprotein, particularly the at least one RNA coding for at least oneantigen comprised by the RNA vaccine, used in the present invention iscodon optimized for the human codon usage. The codons which use for theparticular amino acid the tRNA which occurs the most frequently, e.g.the Gly codon, which uses the tRNA which occurs the most frequently inthe (human) cell, are particularly preferred. In this context, it isparticularly preferable to link the sequential G/C content which isincreased, in particular maximized, in the modified nucleic acidmolecule, particularly of the at least one RNA of the RNA vaccineencoding at least one antigen in the inventive vaccine/inhibitorcombination, with the “frequent” codons without modifying the amino acidsequence of the protein encoded by the coding region of the nucleic acidmolecule. This preferred embodiment allows provision of a particularlyefficiently translated and stabilized (modified) nucleic acid,particularly of the at least one RNA of the RNA vaccine encoding atleast one antigen in the inventive vaccine/inhibitor combination.

Derivative of a nucleic acid molecule: A derivative of a nucleic acidmolecule is defined herein in the same manner as a modified nucleicacid, as defined above.

Nucleotide analogues: Nucleotide analogues are nucleotides structurallysimilar (analogue) to naturally occurring nucleotides which includephosphate backbone modifications, sugar modifications, or modificationsof the nucleobase.

UTR modification: A nucleic acid molecule, especially if the nucleicacid is in the form of a coding nucleic acid molecule, particularly theat least one RNA of the RNA vaccine comprising at least one open readingframe coding for at least one antigen according to the invention,preferably has at least one modified 5′ and/or 3′ UTR sequence (UTRmodification). These in the 5′ and/or 3′ untranslated regions (UTR)included sequences may have the effect of increasing the half-life ofthe nucleic acid in the cytosol or may increase the translation of theencoded protein or peptide. These UTR sequences can have 100% sequenceidentity to naturally occurring sequences which occur in viruses,bacteria and eukaryotes, but can also be partly or completely synthetic.The untranslated sequences (UTR) of the (alpha-) globin gene, e.g. fromHomo sapiens or Xenopus laevis may be mentioned as an example ofstabilizing sequences which can be used for a stabilized nucleic acid.Another example of a stabilizing sequence has the general formula(C/U)CCAN×CCC(U/A)Py×UC(C/U)CC which is contained in the 3′UTR of thevery stable RNA which codes for (alpha-)globin, type(I)-collagen,15-lipoxygenase or for tyrosine hydroxylase (cf. Holcik et al., Proc.Natl. Acad. Sci. USA 1997, 94: 2410 to 2414). Particularly preferred inthe context of the present invention is the mutated UTR of(alpha-)globin comprising the following sequence GCCCGaTGGG CCTCCCAACGGGCCCTCCTC CCCTCCTTGC ACCG (SEQ ID NO. 1) (the underlined nucleotideshows the mutation compared to the wild type sequence), which is alsotermed herein as muag. Such introduced UTR sequences can of course beused individually or in combination with one another and also incombination with other sequence modifications known to a person skilledin the art.

Histone stem-loop: In the context of the present invention, a histonestem-loop sequence is preferably selected from at least one of thefollowing formulae (I) or (II):

formula (I) (stem-loop sequence without stem bordering elements):

formula (II) (stem-loop sequence with stem bordering elements):

wherein:

-   -   stem1 or stem2 bordering elements N1-6 is a consecutive sequence        of 1 to 6, preferably of 2 to 6, more preferably of 2 to 5, even        more preferably of 3 to 5, most preferably of 4 to 5 or 5 N,        wherein each N is independently from another selected from a        nucleotide selected from A, U, T, G and C, or a nucleotide        analogue thereof;    -   stem 1 [N₀₋₂GN₃₋₅] is reverse complementary or partially reverse        complementary with element stem2, and is a consecutive sequence        between of 5 to 7 nucleotides;        -   wherein N₀₋₂ is a consecutive sequence of 0 to 2, preferably            of 0 to 1, more preferably of 1 N, wherein each N is            independently from another selected from a nucleotide            selected from A, U, T, G and C or a nucleotide analogue            thereof;        -   wherein N₃₋₅ is a consecutive sequence of 3 to 5, preferably            of 4 to 5, more preferably of 4 N, wherein each N is            independently from another selected from a nucleotide            selected from A, U, T, G and C or a nucleotide analogue            thereof, and        -   wherein G is guanosine or an analogue thereof, and may be            optionally replaced by a cytidine or an analogue thereof,            provided that its complementary nucleotide cytidine in stem2            is replaced by guanosine;    -   loop sequence [N₀₋₄(U/T)N_(0-4]) is located between elements        stem1 and stem2, and is a consecutive sequence of 3 to 5        nucleotides, more preferably of 4 nucleotides;        -   wherein each N₀₋₄ is independent from another a consecutive            sequence of 0 to 4, preferably of 1 to 3, more preferably of            1 to 2 N, wherein each N is independently from another            selected from a nucleotide selected from A, U, T, G and C or            a nucleotide analogue thereof; and wherein U/T represents            uridine, or optionally thymidine;    -   stem2 [N₃₋₅CN_(0-2]) is reverse complementary or partially        reverse complementary with element stem1, and is a consecutive        sequence between of 5 to 7 nucleotides;        -   wherein N₃₋₅ is a consecutive sequence of 3 to 5, preferably            of 4 to 5, more preferably of 4 N, wherein each N is            independently from another selected from a nucleotide            selected from A, U, T, G and C or a nucleotide analogue            thereof;        -   wherein N₀₋₂ is a consecutive sequence of 0 to 2, preferably            of 0 to 1, more preferably of 1 N, wherein each N is            independently from another selected from a nucleotide            selected from A, U, T, G or C or a nucleotide analogue            thereof; and        -   wherein C is cytidine or an analogue thereof, and may be            optionally replaced by a guanosine or an analogue thereof            provided that its complementary nucleoside guanosine in            stem1 is replaced by cytidine;    -   wherein    -   stem1 and stem2 are capable of base pairing with each other        forming a reverse complementary sequence, wherein base pairing        may occur between stem1 and stem2, e.g. by Watson-Crick base        pairing of nucleotides A and U/T or G and C or by        non-Watson-Crick base pairing e.g. wobble base pairing, reverse        Watson-Crick base pairing, Hoogsteen base pairing, reverse        Hoogsteen base pairing or are capable of base pairing with each        other forming a partially reverse complementary sequence,        wherein an incomplete base pairing may occur between stem1 and        stem2, on the basis that one or more bases in one stem do not        have a complementary base in the reverse complementary sequence        of the other stem.

Nucleic acid synthesis: Nucleic acid molecules used according to theinvention as defined herein may be prepared using any method known inthe art, including synthetic methods such as e.g. solid phase synthesis,in vivo propagation (e.g. in vivo propagation of viruses), as well as invitro methods, such as in vitro transcription reactions.

For preparation of a nucleic acid molecule, especially if the nucleicacid is in the form of an RNA or mRNA, a corresponding DNA molecule maye.g. be transcribed in vitro. This DNA matrix preferably comprises asuitable promoter, e.g. a T7 or SP6 promoter, for in vitrotranscription, which is followed by the desired nucleotide sequencecoding for the nucleic acid molecule, e.g. mRNA, to be prepared and atermination signal for in vitro transcription. The DNA molecule, whichforms the matrix of the at least one RNA of interest, may be prepared byfermentative proliferation and subsequent isolation as part of a plasmidwhich can be replicated in bacteria. Plasmids which may be mentioned assuitable for the present invention are e.g. the plasmids pT7Ts (GenBankaccession number U26404; Lai et al., Development 1995, 121: 2349 to2360), pGEM® series, e.g. pGEM®-1 (GenBank accession number X65300; fromPromega) and pSP64 (GenBank accession number X65327); cf. also Mezei andStorts, Purification of PCR Products, in: Griffin and Griffin (ed.), PCRTechnology: Current Innovation, CRC Press, Boca Raton, Fla., 2001.

RNA: RNA is the usual abbreviation for ribonucleic-acid. It is a nucleicacid molecule, i.e. a polymer consisting of nucleotides. Thesenucleotides are usually adenosine-monophosphate, uridine-monophosphate,guanosine-monophosphate and cytidine-monophosphate monomers which areconnected to each other along a so-called backbone. The backbone isformed by phosphodiester bonds between the sugar, i.e. ribose, of afirst and a phosphate moiety of a second, adjacent monomer. The specificsuccession of the monomers is called the RNA-sequence.

Messenger RNA (mRNA): In eukaryotic cells, transcription is typicallyperformed inside the nucleus or the mitochondria. In vivo, transcriptionof DNA usually results in the so-called premature RNA which has to beprocessed into so-called messenger RNA, usually abbreviated as mRNA.Processing of the premature RNA, e.g. in eukaryotic organisms, comprisesa variety of different posttranscriptional-modifications such assplicing, 5′-capping, polyadenylation, export from the nucleus or themitochondria and the like. The sum of these processes is also calledmaturation of RNA. The mature messenger RNA usually provides thenucleotide sequence that may be translated into an amino acid sequenceof a particular peptide or protein. Typically, a mature mRNA comprises a5′-cap, a 5′UTR, an open reading frame, a 3′UTR and a poly(A) sequence.In the context of the present invention, an mRNA may also be anartificial molecule, i.e. a molecule not occurring in nature. This meansthat the mRNA in the context of the present invention may, e.g.,comprise a combination of a 5′UTR, open reading frame, 3′UTR and poly(A)sequence, which does not occur in this combination in nature.

Retrovirus: A retrovirus is an RNA virus that is duplicated in a hostcell using the reverse transcriptase enzyme to produce DNA from its RNAgenome. The DNA is then incorporated into the host's genome by anintegrase enzyme. The virus thereafter replicates as part of the hostcell's DNA and then undergoes the usual transcription and translationalprocesses to express the genes carried by the virus. Often lentiviruseswere used for gene therapy purposes. For safety reasons lentiviralvectors normally do not carry the genes required for their replication.To produce a lentivirus, several plasmids are transfected into aso-called packaging cell line, commonly HEK 293. One or more plasmids,generally referred to as packaging plasmids, encode the virion proteins,such as the capsid and the reverse transcriptase. Another plasmidcontains the genetic material to be delivered by the vector. It istranscribed to produce the single-stranded RNA viral genome which ispackaged into the virion, which is used for infection of cells for genetherapy purposes or genetic vaccination.

Virion: Virus particles (known as virions) consist of two or threeparts: i) the genetic material (comprising viral genes and optionalsubstituted heterologous genes) made from either DNA or RNA; ii) aprotein coat that protects these genes; and in some cases iii) anenvelope of lipids that surrounds the protein coat when they are outsidea cell.

Self-replicating RNA (Replicons): Self-replicating RNA are deliveryvectors based on alphaviruses which have been developed from SemlikiForest virus (SFV), Sindbis (SIN) virus, and Venezuelan equineencephalitis (VEE) virus. Alphaviruses are single stranded RNA virusesin which heterologous genes of interest may substitute for thealphavirus' structural genes. By providing the structural genes intrans, the replicon RNA is packaged into replicon particles (RP) whichmay be used for gene therapy purposes or genetic vaccination (see forexample Vander Veen et al., 2012. Alphavirus replicon vaccines. AnimalHealth Research Reviews 13(1):1-9). After entry into the host cell, thegenomic viral RNA initially serves as an mRNA for translation of theviral nonstructural proteins (nsPs) required for initiation of viral RNAamplification. RNA replication occurs via synthesis of a full-lengthminus-strand intermediate that is used as the template for synthesis ofadditional genome-length RNAs and for transcription of a plus-strandsubgenomic RNA from an internal promoter. Such RNA may then beconsidered as self-replicating RNA, since the non-structural proteinsresponsible for replication (and transcription of the heterologousgenes) are still present in such replicon. Such alphavirus vectors arereferred to as “replicons.”

Replicon particle: A replicon particle consist of two or three parts: i)the genetic material (=the replicon) (comprising viral genes andoptional substituted heterologous genes) made from either DNA or RNA;ii) a protein coat that protects these genes; and in some cases iii) anenvelope of lipids that surrounds the protein coat when they are outsidea cell.

Isolated RNA: Isolated RNA is defined herein as RNA which is not part ofa cell, an irradiated cell or a cell lysate. An isolated RNA may beproduced by isolation and/or purification from cells or cell lysates, orfrom in vitro transcription systems.

The term isolated RNA in the context of the present invention is definedherein as RNA which is not part of cells, irradiated cells or celllysates including the RNA of the RNA vaccine or which is not part ofcells transfected with the RNA of the RNA vaccine. In other words, theterm “isolated RNA” excludes (ex vivo) transfected or modulated cellsused as RNA vaccine, in particular excludes ex vivo transfected ormodulated immune cells, such as dendritic cells (DC), e.g. DCtransfected/transduced with an RNA used as RNA vaccine. Furthermore, theterm isolated RNA excludes RNA comprised in cells, irradiated cells orcell lysate comprising naturally the at least one RNA coding for anantigen used as RNA vaccine. Accordingly, the RNA vaccine of theinventive combination does preferably not comprise modulated ortransfected cells, in particular no transfected or modulated immunecells (e.g. antigen presenting cells), more particularly no transfectedor modulated DC, irradiated cells or cell lysates comprising naturallythe at least one RNA of the RNA vaccine. By that embodiment, it becomesclear that the RNA vaccine used in the present invention does preferablynot correspond to a cell-based vaccine or a DC-based vaccine, or, moregenerally, does preferably not correspond to a cell-based vaccine or,more generally, the RNA vaccine used in the present invention ispreferably free of cells and the inventive combination to beadministered provides the RNA vaccine as RNA, more preferably as mRNA,optionally in association with a carrier and/or adjuvant component. Theterm isolated RNA also includes RNA that is complexed with furthercomponents e.g. peptides, proteins, carriers etc., RNA packaged inparticles like e.g. replicon particles or virus particles (virions) andRNA contained in solution which may additional to the RNA comprisefurther components e.g. buffer, stabilization reagents, RNAseinhibitors, etc.

Sequence of a nucleic acid molecule: The sequence of a nucleic acidmolecule is typically understood to be the particular and individualorder, i.e. the succession of its nucleotides.

Sequence of a protein or peptide: The sequence of a protein or peptideis typically understood to be the order, i.e. the succession of itsamino acids.

Sequence identity: Two or more sequences are identical if they exhibitthe same length and order of nucleotides or amino acids. The percentageof identity typically describes the extent to which two sequences areidentical, i.e. it typically describes the percentage of nucleotidesthat correspond in their sequence position with identical nucleotides ofa reference-sequence. For determination of the degree of identity, thesequences to be compared are considered to exhibit the same length, i.e.the length of the longest sequence of the sequences to be compared. Thismeans that a first sequence consisting of 8 nucleotides is 80% identicalto a second sequence consisting of 10 nucleotides comprising the firstsequence. In other words, in the context of the present invention,identity of sequences preferably relates to the percentage ofnucleotides of a sequence which have the same position in two or moresequences having the same length. Gaps are usually regarded asnon-identical positions, irrespective of their actual position in analignment.

Fragment of a sequence: a fragment of a sequence is typically a shorterportion of a full-length sequence of e.g. a nucleic acid sequence or anamino acid sequence. Accordingly, a fragment of a sequence, typically,consists of a sequence that is identical to the corresponding stretch orcorresponding stretches within the full-length sequence. A preferredfragment of a sequence in the context of the present invention, consistsof a continuous stretch of entities, such as nucleotides or amino acids,corresponding to a continuous stretch of entities in the molecule thefragment is derived from, which represents at least 5%, preferably atleast 20%, preferably at least 30%, more preferably at least 40%, morepreferably at least 50%, even more preferably at least 60%, even morepreferably at least 70%, and most preferably at least 80% of the total(i.e. full-length) molecule from which the fragment is derived. Thus,for example, a fragment of a protein or peptide antigen preferablycorresponds to a continuous stretch of entities in the protein orpeptide antigen the fragment is derived from, which represents at least5%, preferably at least 20%, preferably at least 30%, more preferably atleast 40%, more preferably at least 50%, even more preferably at least60%, even more preferably at least 70%, and most preferably at least 80%of the total (i.e. full-length) protein or peptide antigen. It isparticularly preferred that the fragment of a sequence is a functionalfragment, i.e. that the fragment fulfils one or more of the functionsfulfilled by the sequence the fragment is derived from. For example, afragment of a protein or peptide antigen preferably exhibits at leastone antigenic function (e.g. is capable of eliciting a specific immunereaction against at least one antigen determinant in said protein orpeptide antigen) of the protein or peptide antigen the fragment isderived from.

Fragments of proteins: “Fragments” of proteins or peptides, i.e.,fragments of amino acid sequences, in the context of the presentinvention may, typically, comprise a sequence of a protein or peptide asdefined herein, which is, with regard to its amino acid sequence (or itsencoding nucleic acid molecule), N-terminally, C-terminally and/orintrasequentially truncated compared to the amino acid sequence of theoriginal (native) protein (or its encoded nucleic acid molecule). Suchtruncation may thus occur either on the amino acid level orcorrespondingly on the nucleic acid level. A sequence identity withrespect to such a fragment as defined herein may therefore preferablyrefer to the entire protein or peptide as defined herein or to theentire (coding) nucleic acid molecule of such a protein or peptide.

Likewise, “fragments” of nucleic acid sequences in the context of thepresent invention may comprise a sequence of a nucleic acid as definedherein, which is, with regard to its nucleic acid molecule 5′-, 3′-and/or intrasequentially truncated compared to the nucleic acid moleculeof the original (native) nucleic acid molecule. A sequence identity withrespect to such a fragment as defined herein may therefore preferablyrefer to the entire nucleic acid as defined herein.

Transfection: The term ‘transfection’ refers to the introduction ofnucleic acid molecules, such as DNA or RNA (e.g. mRNA) molecules, intocells, preferably into eukaryotic cells. In the context of the presentinvention, the term ‘transfection’ encompasses any method known to theskilled person for introducing nucleic acid molecules, preferably RNAmolecules into cells, preferably into eukaryotic cells, such as intomammalian cells. Such methods encompass, for example, electroporation,lipofection, e.g. based on cationic lipids and/or liposomes, calciumphosphate precipitation, nanoparticle based transfection, virus basedtransfection, or transfection based on cationic polymers, such asDEAE-dextran or polyethylenimine etc.

Carrier: A carrier in the context of the invention may typically be acompound that facilitates transport and/or complexation of anothercompound (cargo). A carrier may be associated to its cargo by covalentor non-covalent interaction. A carrier may transport nucleic acids, e.g.RNA or DNA, to the target cells. The carrier may—for some embodiments—bea cationic or polycationic compound or a polymeric carrier as definedherein. A carrier, in the context of the present invention, ispreferably suitable as carrier for nucleic acid molecules, e.g. formediating dissolution in physiological acceptable liquids, transport andcellular uptake of the nucleic acid molecules or a vector. Accordingly,a carrier, in the context of the present invention, may be a componentwhich may be suitable for depot and delivery of a nucleic acid moleculeor vector. Such carriers may be, for example, cationic or polycationiccarriers or compounds which may serve as transfection or complexationagent. Particularly preferred carriers or polymeric carriers in thiscontext are cationic or polycationic compounds.

Cationic or Polycationic Compound/Component:

The term “cationic or polycationic compound/component” typically refersto a charged molecule, which is positively charged (cation) at a pHvalue typically from 1 to 9, preferably at a pH value of or below 9(e.g. from 5 to 9), of or below 8 (e.g. from 5 to 8), of or below 7(e.g. from 5 to 7), most preferably at a physiological pH, e.g. from 7.3to 7.4. Accordingly, a cationic or polycationic compound/component maybe any positively charged compound or polymer, preferably a cationic orpolycationic peptide or protein which is positively charged underphysiological conditions, particularly under physiological conditions invivo. A ‘cationic peptide or protein’ may contain at least onepositively charged amino acid, or more than one positively charged aminoacid, e.g. selected from Arg, His, Lys or Orn. Accordingly,‘polycationic’ compounds are also within the scope exhibiting more thanone positive charge under the conditions given. In this context acationic peptide or protein contains a larger number of cationic aminoacids, e.g. a larger number of Arg, His, Lys or Orn, than negativelycharged amino acids. In a preferred embodiment, a cationic peptide orprotein in the context of the present invention contains a larger numberof cationic amino acids, e.g. a larger number of Arg, His, Lys or Orn,than other residues.

The term “cationic or polycationic compound” in the context of thepresent invention preferably refers to compounds which can be used astransfection or complexation agent, particularly of nucleic acids, usedaccording to the invention.

Cationic or polycationic compounds according to the invention, beingparticularly preferred agents in this context include protamine,nucleoline, spermine or spermidine, or other cationic peptides orproteins, such as poly-L-lysine (PLL), poly-arginine, basicpolypeptides, cell penetrating peptides (CPPs), including HIV-bindingpeptides, HIV-1 Tat (HIV), Tat-derived peptides, Penetratin, VP22derived or analog peptides, HSV VP22 (Herpes simplex), MAP, KALA orprotein transduction domains (PTDs), PpT620, prolin-rich peptides,arginine-rich peptides, lysine-rich peptides, MPG-peptide(s), Pep-1,L-oligomers, Calcitonin peptide(s), Antennapedia-derived peptides(particularly from Drosophila antennapedia), pAntp, pIsl, FGF,Lactoferrin, Transportan, Buforin-2, Bac715-24, SynB, SynB(1), pVEC,hCT-derived peptides, SAP, or histones. Protamine is particularlypreferred.

Additionally, preferred cationic or polycationic proteins or peptidesused as transfection or complexation agent may be selected from thefollowing proteins or peptides having the following total formula (III):

(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x),   (formula (III))

wherein l+m+n+o+x=8-15, and l, m, n or o independently of each other maybe any number selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14 or 15, provided that the overall content of Arg, Lys, His and Ornrepresents at least 50% of all amino acids of the oligopeptide; and Xaamay be any amino acid selected from native (=naturally occurring) ornon-native amino acids except of Arg, Lys, His or Orn; and x may be anynumber selected from 0, 1, 2, 3 or 4, provided, that the overall contentof Xaa does not exceed 50% of all amino acids of the oligopeptide.Particularly preferred cationic peptides in this context are e.g. Arg₇,Arg₈, Arg₉, H₃R₉, R₉H₃, H₃R₉H₃, YSSR₉SSY, (RKH)4, Y(RKH)₂R, etc.

Further preferred cationic or polycationic compounds, which can be usedas transfection or complexation agent may include cationicpolysaccharides, for example chitosan, polybrene, cationic polymers,e.g. polyethyleneimine (PEI), cationic lipids, e.g. DOTMA:[1-(2,3-sioleyloxy)propyl)]-N,N,N-trimethylammonium chloride, DMRIE,di-C14-amidine, DOTIM, SAINT, DC-Chol, BGTC, CTAP, DOPC, DODAP, DOPE:Dioleyl phosphatidylethanol-amine, DOSPA, DODAB, DOIC, DMEPC, DOGS:Dioctadecylamidoglicylspermin, DIMRI: Dimyristo-oxypropyl dimethylhydroxyethyl ammonium bromide, DOTAP:dioleoyloxy-3-(trimethylammonio)propane, DC-6-14:O,O-ditetradecanoyl-N-(α-trimethylammonioacetyl)diethanolamine chloride,CLIP1: rac-[(2,3-dioctadecyloxypropyl)(2-hydroxyethyl)]-dimethylammoniumchloride, CLIP6:rac-[2(2,3-dihexadecyloxypropyl-oxymethyloxy)ethyl]trimethylammonium,CLIP9:rac-[2(2,3-dihexadecyloxypropyl-oxysuccinyloxy)ethyl]-trimethylammonium,oligofectamine, Lipofectamine® or cationic or polycationic polymers,e.g. modified polyaminoacids, such as β-aminoacid-polymers or reversedpolyamides, etc., modified polyethylenes, such as PVP(poly(N-ethyl-4-vinylpyridinium bromide)), etc., modified acrylates,such as pDMAEMA (poly(dimethylaminoethyl methylacrylate)), etc.,modified amidoamines such as pAMAM (poly(amidoamine)), etc., modifiedpolybetaaminoester (PBAE), such as diamine end modified 1,4 butanedioldiacrylate-co-5-amino-1-pentanol polymers, etc., dendrimers, such aspolypropylamine dendrimers or pAMAM based dendrimers, etc.,polyimine(s), such as PEI: poly(ethyleneimine), poly(propyleneimine),etc., polyallylamine, sugar backbone based polymers, such ascyclodextrin based polymers, dextran based polymers, chitosan, etc.,silan backbone based polymers, such as PMOXA-PDMS copolymers, etc.,blockpolymers consisting of a combination of one or more cationic blocks(e.g. selected from a cationic polymer as mentioned above) and of one ormore hydrophilic or hydrophobic blocks (e.g. polyethyleneglycole); etc.

Polymeric carrier: A polymeric carrier is typically a carrier that isformed of a polymer. A polymeric carrier in the context of the presentinvention used as transfection or complexation agent might be apolymeric carrier formed by disulfide-crosslinked cationic components.The disulfide-crosslinked cationic components may be the same ordifferent from each other. The polymeric carrier can also containfurther components. It is also particularly preferred that the polymericcarrier comprises mixtures of cationic peptides, proteins or polymersand optionally further components as defined herein, which arecrosslinked by disulfide bonds as described herein.

In this context the cationic components, which form basis for thepolymeric carrier by disulfide-crosslinkage, are typically selected fromany suitable cationic or polycationic peptide, protein or polymersuitable for this purpose, particular any cationic or polycationicpeptide, protein or polymer capable to complex a nucleic acid as definedaccording to the present invention, and thereby preferably condensingthe nucleic acid. The cationic or polycationic peptide, protein orpolymer, is preferably a linear molecule, however, branched cationic orpolycationic peptides, proteins or polymers may also be used.

Every disulfide-crosslinking cationic or polycationic protein, peptideor polymer of the polymeric carrier, which may be used to complex e.g.the at least one RNA of the RNA vaccine encoding at least one antigen oran adjuvant nucleic acid in the inventive vaccine/inhibitor combinationcontains at least one —SH moiety, most preferably at least one cysteineresidue or any further chemical group exhibiting an —SH moiety, capableto form a disulfide linkage upon condensation with at least one furthercationic or polycationic protein, peptide or polymer as cationiccomponent of the polymeric carrier as mentioned herein.

As defined above, the polymeric carrier, which may be used to complexe.g. the at least one RNA of the RNA vaccine encoding at least oneantigen or an adjuvant nucleic acid in the inventive vaccine/inhibitorcombination may be formed by disulfide-crosslinked cationic (orpolycationic) components.

According to one first alternative, at least one cationic (orpolycationic) component of the polymeric carrier, which may be used inthis context may be selected from cationic or polycationic peptides orproteins. Such cationic or polycationic peptides or proteins preferablyexhibit a length of about 3 to 100 amino acids, preferably a length ofabout 3 to 50 amino acids, more preferably a length of about 3 to 25amino acids, e.g. a length of about 3 to 10, 5 to 15, 10 to 20 or 15 to25 amino acids. Alternatively or additionally, such cationic orpolycationic peptides or proteins may exhibit a molecular weight ofabout 0.01 kDa to about 100 kDa, including a molecular weight of about0.5 kDa to about 100 kDa, preferably of about 10 kDa to about 50 kDa,even more preferably of about 10 kDa to about 30 kDa.

In the specific case that the cationic component of the polymericcarrier, which may be used to complex e.g. the at least one RNA of theRNA vaccine encoding at least one antigen or an adjuvant nucleic acid inthe inventive vaccine/inhibitor combination comprises a cationic orpolycationic peptide or protein, the cationic properties of the cationicor polycationic peptide or protein or of the entire polymeric carrier,if the polymeric carrier is entirely composed of cationic orpolycationic peptides or proteins, may be determined upon its content ofcationic amino acids. Preferably, the content of cationic amino acids inthe cationic or polycationic peptide or protein and/or the polymericcarrier is at least 10%, 20%, or 30%, preferably at least 40%, morepreferably at least 50%, 60% or 70%, but also preferably at least 80%,90%, or even 95%, 96%, 97%, 98%, 99% or 100%, most preferably at least30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, ormay be in the range of about 10% to 90%, more preferably in the range ofabout 15% to 75%, even more preferably in the range of about 20% to 50%,e.g. 20, 30, 40 or 50%, or in a range formed by any two of the aforementioned values, provided, that the content of all amino acids, e.g.cationic, lipophilic, hydrophilic, aromatic and further amino acids, inthe cationic or polycationic peptide or protein, or in the entirepolymeric carrier, if the polymeric carrier is entirely composed ofcationic or polycationic peptides or proteins, is 100%.

Preferably, such cationic or polycationic peptides or proteins of thepolymeric carrier, which comprise or are additionally modified tocomprise at least one —SH moiety, are selected from, without beingrestricted thereto, cationic peptides or proteins such as protamine,nucleoline, spermine or spermidine, oligo- or poly-L-lysine (PLL), basicpolypeptides, oligo or poly-arginine, cell penetrating peptides (CPPs),chimeric CPPs, such as Transportan, or MPG peptides, HIV-bindingpeptides, Tat, HIV-1 Tat (HIV), Tat-derived peptides, members of thepenetratin family, e.g. Penetratin, Antennapedia-derived peptides(particularly from Drosophila antennapedia), pAntp, pIsl, etc.,antimicrobial-derived CPPs e.g. Buforin-2, Bac715-24, SynB, SynB(1),pVEC, hCT-derived peptides, SAP, MAP, PpTG20, Loligomere, FGF,Lactoferrin, histones, VP22 derived or analog peptides, Pestivirus Erns,HSV, VP22 (Herpes simplex), MAP, KALA or protein transduction domains(PTDs, PpT620, prolin-rich peptides, arginine-rich peptides, lysine-richpeptides, Pep-1, L-oligomers, Calcitonin peptide(s), etc.

Alternatively or additionally, such cationic or polycationic peptides orproteins of the polymeric carrier, which comprise or are additionallymodified to comprise at least one —SH moiety, are selected from, withoutbeing restricted thereto, following cationic peptides having thefollowing sum formula (IV):

{(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x)};   formula (IV)

wherein l+m+n+o+x=3-100, and l, m, n or o independently of each other isany number selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21-30, 31-40, 41-50, 51-60, 61-70, 71-80,81-90 and 91-100 provided that the overall content of Arg (Arginine),Lys (Lysine), His (Histidine) and Orn (Ornithine) represents at least10% of all amino acids of the oligopeptide; and Xaa is any amino acidselected from native (=naturally occurring) or non-native amino acidsexcept of Arg, Lys, His or Orn; and x is any number selected from 0, 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21-30, 31-40, 41-50, 51-60, 61-70, 71-80, 81-90, provided, that theoverall content of Xaa does not exceed 90% of all amino acids of theoligopeptide. Any of amino acids Arg, Lys, His, Orn and Xaa may bepositioned at any place of the peptide. In this context cationicpeptides or proteins in the range of 7-30 amino acids are particularpreferred. Even more preferred peptides of this formula areoligoarginines such as e.g. Arg₇, Arg₈, Arg₉, Arg₁₂, His₃Arg₉, Arg₉His₃,His₃Arg₉His₃, His₆Arg₉His₆, His₃Arg₄His₃, His₆Arg₄His₆,TyrSer₂Arg₉Ser₂Tyr, (ArgLysHis)₄, Tyr(ArgLysHis)₂Arg, etc.

According to a one further particular preferred embodiment, the cationicor polycationic peptide or protein of the polymeric carrier, whendefined according to formula{(Arg)_(l);(Lys)_(m);(His)_(n)(Orn)_(o);(Xaa)_(x)} (formula (IV)) asshown above and which comprises or is additionally modified to compriseat least one —SH moiety, may be, without being restricted thereto,selected from subformula (IVa):

{(Arg)_(l);(Lys)_(m),(His)_(n);(Orn)_(o);(Xaa′)_(x)(Cys)_(y)}   formula(IVa)

wherein (Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o); and x are as definedherein, Xaa′ is any amino acid selected from native (=naturallyoccurring) or non-native amino acids except of Arg, Lys,

His, Orn or Cys and y is any number selected from 0, 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21-30, 31-40,41-50, 51-60, 61-70, 71-80 and 81-90, provided that the overall contentof Arg (Arginine), Lys (Lysine), His (Histidine) and Orn (Ornithine)represents at least 10% of all amino acids of the oligopeptide.

This embodiment may apply to situations, wherein the cationic orpolycationic peptide or protein of the polymeric carrier, e.g. whendefined according to empirical formula(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x) (formula (IV)) asshown above, comprises or has been modified with at least one cysteineas —SH moiety in the above meaning such that the cationic orpolycationic peptide as cationic component carries at least onecysteine, which is capable to form a disulfide bond with othercomponents of the polymeric carrier.

According to another particular preferred embodiment, the cationic orpolycationic peptide or protein of the polymeric carrier, when definedaccording to formula {(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x)}(formula (IV)) as shown above, may be, without being restricted thereto,selected from subformula (IVb):

Cys¹ {(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x)} Cys²   formula(IVb)

wherein empirical formula{(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x)} (formula (IV)) is asdefined herein and forms a core of an amino acid sequence according to(semiempirical) formula (IV) and wherein Cys¹ and Cys² are Cysteinesproximal to, or terminal to(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x). This embodiment mayapply to situations, wherein the cationic or polycationic peptide orprotein of the polymeric carrier, which may be used to complex the atleast one RNA of the RNA vaccine encoding at least one antigen or anadjuvant nucleic acid in the inventive vaccine/inhibitor combination,e.g. when defined according to empirical formula(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x) (formula (IV)) asshown above, has been modified with at least two cysteines as —SHmoieties in the above meaning such that the cationic or polycationicpeptide of the inventive polymeric carrier carries at least two(terminal) cysteines, which are capable to form a disulfide bond withother components of the polymeric carrier.

According to a second alternative, at least one cationic (orpolycationic) component of the polymeric carrier may be selected frome.g. any (non-peptidic) cationic or polycationic polymer suitable inthis context, provided that this (non-peptidic) cationic or polycationicpolymer exhibits or is modified to exhibit at least one —SH-moiety,which provide for a disulfide bond linking the cationic or polycationicpolymer with another component of the polymeric carrier as definedherein. Thus, likewise as defined herein, the polymeric carrier maycomprise the same or different cationic or polycationic polymers.

In the specific case that the cationic component of the polymericcarrier comprises a (non-peptidic) cationic or polycationic polymer thecationic properties of the (non-peptidic) cationic or polycationicpolymer may be determined upon its content of cationic charges whencompared to the overall charges of the components of the cationicpolymer. Preferably, the content of cationic charges in the cationicpolymer at a (physiological) pH as defined herein is at least 10%, 20%,or 30%, preferably at least 40%, more preferably at least 50%, 60% or70%, but also preferably at least 80%, 90%, or even 95%, 96%, 97%, 98%,99% or 100%, most preferably at least 30%, 40%, 50%, 60%, 70%, 80%, 90%,95%, 96%, 97%, 98%, 99% or 100%, or may be in the range of about 10% to90%, more preferably in the range of about 30% to 100%, even preferablyin the range of about 50% to 100%, e.g. 50, 60, 70, 80%, 90% or 100%, orin a range formed by any two of the afore mentioned values, provided,that the content of all charges, e.g. positive and negative charges at a(physiological) pH as defined herein, in the entire cationic polymer is100%.

Preferably, the (non-peptidic) cationic component of the polymericcarrier represents a cationic or polycationic polymer, typicallyexhibiting a molecular weight of about 0.1 or 0.5 kDa to about 100 kDa,preferably of about 1 kDa to about 75 kDa, more preferably of about 5kDa to about 50 kDa, even more preferably of about 5 kDa to about 30kDa, or a molecular weight of about 10 kDa to about 50 kDa, even morepreferably of about 10 kDa to about 30 kDa. Additionally, the(non-peptidic) cationic or polycationic polymer typically exhibits atleast one —SH-moiety, which is capable to form a disulfide linkage uponcondensation with either other cationic components or other componentsof the polymeric carrier as defined herein.

In the above context, the (non-peptidic) cationic component of thepolymeric carrier, which may be used to complex e.g. the at least oneRNA of the RNA vaccine encoding at least one antigen or an adjuvantnucleic acid in the inventive vaccine/inhibitor combination may beselected from acrylates, modified acrylates, such as pDMAEMA(poly(dimethylaminoethyl methylacrylate)), chitosanes, aziridines or2-ethyl-2-oxazoline (forming oligo ethylenimines or modifiedoligoethylenimines), polymers obtained by reaction of bisacrylates withamines forming oligo beta aminoesters or poly amido amines, or otherpolymers like polyesters, polycarbonates, etc. Each molecule of these(non-peptidic) cationic or polycationic polymers typically exhibits atleast one —SH-moiety, wherein these at least one —SH-moiety may beintroduced into the (non-peptidic) cationic or polycationic polymer bychemical modifications, e.g. using imonothiolan, 3-thio propionic acidor introduction of —SH-moieties containing amino acids, such as cysteineor any further (modified) amino acid. Such —SH-moieties are preferablyas already defined above.

According to a particularly preferred embodiment, the further component,which may be contained in the polymeric carrier, which may be used tomodify the different (short) cationic or polycationic peptides or(non-peptidic) polymers forming basis for the polymeric carrier or thebiophysical/biochemical properties of the polymeric carrier as definedherein, is an amino acid component (AA). According to the presentinvention, the amino acid component (AA) comprises a number of aminoacids preferably in a range of about 1 to 100, preferably in a range ofabout 1 to 50, more preferably selected from a number comprising 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15-20, or may be selectedfrom a range formed by any two of the afore mentioned values. In thiscontext the amino acids of amino acid component (AA) can be chosenindependently from each other. For example if in the polymeric carriertwo or more (AA) components are present they can be the same or can bedifferent from each other.

The amino acid component (AA) may contain or may be flanked (e.g.terminally) by a —SH containing moiety, which allows introducing thiscomponent (AA) via a disulfide bond into the polymeric carrier asdefined herein. In the specific case that the —SH containing moietyrepresents a cysteine, the amino acid component (AA) may also be read as-Cys-(AA)-Cys- wherein Cys represents cysteine and provides for thenecessary —SH-moiety for a disulfide bond. The —SH containing moiety maybe also introduced into amino acid component (AA) using any ofmodifications or reactions as shown above for the cationic component orany of its components.

Furthermore, the amino acid component (AA) may be provided with two—SH-moieties (or even more), e.g. in a form represented by formulaHS-(AA)-SH to allow binding to two functionalities via disulfide bonds,e.g. if the amino acid component (AA) is used as a linker between twofurther components (e.g. as a linker between two cationic polymers).

Alternatively, the amino acid component (AA) may be provided with otherfunctionalities as already described above for the other components ofthe polymeric carrier, which allow binding of the amino acid component(AA) to any of components of the polymeric carrier.

Thus, according to the present invention, the amino acid component (AA)of the polymeric carrier may be bound to further components of thepolymeric carrier, which may be used to complex e.g. the at least oneRNA of the RNA vaccine encoding at least one antigen or an adjuvantnucleic acid in the inventive vaccine/inhibitor with or without using adisulfide linkage.

According to a further and particularly preferred alternative, the aminoacid component (AA), may be used to modify the polymeric carrier,particularly the content of cationic components in the polymeric carrieras defined above.

In the context of the present invention, the amino acid component (AA)may be selected from the following alternatives: an aromatic amino acidcomponent, a hydrophilic (and preferably non charged polar) amino acidcomponent, a lipophilic amino acid component, or a weak basic amino acidcomponent.

According to a further alternative, the amino acid component (AA) may bea signal peptide or signal sequence, a localisation signal or sequence,a nuclear localisation signal or sequence (NLS), an antibody, a cellpenetrating peptide (e.g. TAT), etc. Additionally, according to anotheralternative, the amino acid component (AA) may be a functional peptideor protein, which may modulate the functionality of the polymericcarrier accordingly. Such functional peptides or proteins as the aminoacid component (AA) preferably comprise any peptides or proteins asdefined herein, e.g. as defined herein as antigens. According to onealternative, such further functional peptides or proteins may compriseso called cell penetrating peptides (CPPs) or cationic peptides fortransportation.

According to a last alternative, the amino acid component (AA) mayconsist of or may comprise any peptide or protein which can execute anyfavourable function in the cell.

Particularly preferred are peptides or proteins selected fromtherapeutically active proteins or peptides, from antigens, e.g. tumourantigens, pathogenic antigens (e.g. animal antigens, viral antigens,protozoan antigens, bacterial antigens), from antibodies, fromimmunostimulatory proteins or peptides, from antigen-specific T cellreceptors, or from any other protein or peptide suitable for a specific(therapeutic) application. Particularly preferred are peptide epitopesfrom those antigen(s) encoded by the at least one RNA of the RNA vaccineencoding at least one antigen in the inventive vaccine/inhibitorcombination.

The polymeric carrier, which may be used to complex e.g. the at leastone RNA of the RNA vaccine encoding at least one antigen or an adjuvantnucleic acid in the inventive vaccine/inhibitor combination may compriseat least one of the above mentioned cationic or polycationic peptides,proteins or polymers or further components, e.g. (AA), wherein any ofthe above alternatives may be combined with each other, and may beformed by polymerizing same in a polymerization condensation reactionvia their —SH-moieties.

Further, the polymeric carrier may be selected from a polymeric carriermolecule according to generic formula (V):

L-P¹—S—[S—P²—S]_(n)—S—P³-L   formula (V)

wherein,

-   -   P¹ and P³ are different or identical to each other and represent        a linear or branched hydrophilic polymer chain, each P¹ and P³        exhibiting at least one —SH-moiety, capable to form a disulfide        linkage upon condensation with component P², or alternatively        with (AA), (AA)_(x), or [(AA)_(x)]_(z) if such components are        used as a linker between P¹ and P² or P³ and P²) and/or with        further components (e.g. (AA), (AA)_(x), [(AA)_(x)]_(z) or L),        the linear or branched hydrophilic polymer chain selected        independent from each other from polyethylene glycol (PEG),        poly-N-(2-hydroxypropyl)methacrylamide,        poly-2-(methacryloyloxy)ethyl phosphorylcholines,        poly(hydroxyalkyl L-asparagine), poly(2-(methacryloyloxy)ethyl        phosphorylcholine), hydroxyethyl starch or poly(hydroxyalkyl        L-glutamine), wherein the hydrophilic polymer chain exhibits a        molecular weight of about 1 kDa to about 100 kDa, preferably of        about 2 kDa to about 25 kDa; or more preferably of about 2 kDa        to about 10 kDa, e.g. about 5 kDa to about 25 kDa or 5 kDa to        about 10 kDa;    -   P² is a cationic or polycationic peptide or protein, e.g. as        defined above for the polymeric carrier formed by        disulfide-crosslinked cationic components, and preferably having        a length of about 3 to about 100 amino acids, more preferably        having a length of about 3 to about 50 amino acids, even more        preferably having a length of about 3 to about 25 amino acids,        e.g. a length of about 3 to 10, 5 to 15, 10 to 20 or 15 to 25        amino acids, more preferably a length of about 5 to about 20 and        even more preferably a length of about 10 to about 20; or        -   is a cationic or polycationic polymer, e.g. as defined above            for the polymeric carrier formed by disulfide-crosslinked            cationic components, typically having a molecular weight of            about 0.5 kDa to about 30 kDa, including a molecular weight            of about 1 kDa to about 20 kDa, even more preferably of            about 1.5 kDa to about 10 kDa, or having a molecular weight            of about 0.5 kDa to about 100 kDa, including a molecular            weight of about 10 kDa to about 50 kDa, even more preferably            of about 10 kDa to about 30 kDa;        -   each P² exhibiting at least two —SH-moieties, capable to            form a disulfide linkage upon condensation with further            components P² or component(s) P¹ and/or P³ or alternatively            with further components (e.g. (AA), (AA)_(x), or            [(AA)_(x)]_(z));    -   —S—S— is a (reversible) disulfide bond (the brackets are omitted        for better readability), wherein S preferably represents sulphur        or a —SH carrying moiety, which has formed a (reversible)        disulfide bond. The (reversible) disulfide bond is preferably        formed by condensation of —SH-moieties of either components P¹        and P², P² and P², or P² and P³, or optionally of further        components as defined herein (e.g. L, (AA), (AA)_(x), [(AA)_(z),        etc); The —SH-moiety may be part of the structure of these        components or added by a modification as defined below;    -   is an optional ligand, which may be present or not, and may be        selected independent from the other from RGD, Transferrin,        Folate, a signal peptide or signal sequence, a localization        signal or sequence, a nuclear localization signal or sequence        (NLS), an antibody, a cell penetrating peptide, (e.g. TAT or        KALA), a ligand of a receptor (e.g. cytokines, hormones, growth        factors etc), small molecules (e.g. carbohydrates like mannose        or galactose or synthetic ligands), small molecule agonists,        inhibitors or antagonists of receptors (e.g. RGD peptidomimetic        analogues), or any further protein as defined herein, etc.;    -   n is an integer, typically selected from a range of about 1 to        50, preferably from a range of about 1, 2 or 3 to 30, more        preferably from a range of about 1, 2, 3, 4, or 5 to 25, or a        range of about 1, 2, 3, 4, or 5 to 20, or a range of about 1, 2,        3, 4, or 5 to 15, or a range of about 1, 2, 3, 4, or 5 to 10,        including e.g. a range of about 4 to 9, 4 to 10, 3 to 20, 4 to        20, 5 to 20, or 10 to 20, or a range of about 3 to 15, 4 to 15,        5 to 15, or 10 to 15, or a range of about 6 to 11 or 7 to 10.        Most preferably, n is in a range of about 1, 2, 3, 4, or 5 to        10, more preferably in a range of about 1, 2, 3, or 4 to 9, in a        range of about 1, 2, 3, or 4 to 8, or in a range of about 1, 2,        or 3 to 7.

Each of hydrophilic polymers P¹ and P³ typically exhibits at least one—SH-moiety, wherein the at least one —SH-moiety is capable to form adisulfide linkage upon reaction with component P² or with component (AA)or (AA)_(x), if used as linker between P¹ and P² or P³ and P² as definedbelow and optionally with a further component, e.g. L and/or (AA) or(AA)_(x), e.g. if two or more —SH-moieties are contained. The followingsubformulae “P¹—S—S—P²” and “P²—S—S—P³” within generic formula (V) above(the brackets are omitted for better readability), wherein any of S, P¹and P³ are as defined herein, typically represent a situation, whereinone-SH-moiety of hydrophilic polymers P^(l) and P³ was condensed withone —SH-moiety of component P² of generic formula (V) above, whereinboth sulphurs of these —SH-moieties form a disulfide bond —S—S— asdefined herein in formula (V). These —SH-moieties are typically providedby each of the hydrophilic polymers P¹ and P³, e.g. via an internalcysteine or any further (modified) amino acid or compound which carriesa —SH moiety. Accordingly, the subformulae “P¹—S—S—P²” and “P²—S—S—P³”may also be written as “P¹-Cys-Cys-P²” and “P²-Cys-Cys-P³”, if the—SH-moiety is provided by a cysteine, wherein the term Cys-Cysrepresents two cysteines coupled via a disulfide bond, not via a peptidebond. In this case, the term “—S—S—” in these formulae may also bewritten as “—S-Cys”, as “-Cys-S” or as “-Cys-Cys-”. In this context, theterm “-Cys-Cys-” does not represent a peptide bond but a linkage of twocysteines via their —SH-moieties to form a disulfide bond. Accordingly,the term “-Cys-Cys-” also may be understood generally as“-(Cys-S)-(S-Cys)-”, wherein in this specific case S indicates thesulphur of the —SH-moiety of cysteine. Likewise, the terms “—S-Cys” and“-Cys-S” indicate a disulfide bond between a —SH containing moiety and acysteine, which may also be written as “—S—(S-Cys)” and “-(Cys-S)—S”.Alternatively, the hydrophilic polymers P¹ and P³ may be modified with a—SH moiety, preferably via a chemical reaction with a compound carryinga —SH moiety, such that each of the hydrophilic polymers P¹ and P³carries at least one such —SH moiety. Such a compound carrying a —SHmoiety may be e.g. an (additional) cysteine or any further (modified)amino acid, which carries a —SH moiety. Such a compound may also be anynon-amino compound or moiety, which contains or allows to introduce a—SH moiety into hydrophilic polymers P¹ and P³ as defined herein. Suchnon-amino compounds may be attached to the hydrophilic polymers P¹ andP³ of formula (VI) of the polymeric carrier according to the presentinvention via chemical reactions or binding of compounds, e.g. bybinding of a 3-thio propionic acid or thioimolane, by amide formation(e.g. carboxylic acids, sulphonic acids, amines, etc), by Michaeladdition (e.g. maleinimide moieties, □, □ unsatured carbonyls, etc), byclick chemistry (e.g. azides or alkines), by alkene/alkine methatesis(e.g. alkenes or alkines), imine or hydrozone formation (aldehydes orketons, hydrazins, hydroxylamins, amines), complexation reactions(avidin, biotin, protein G) or components which allow S_(n)-typesubstitution reactions (e.g. halogenalkans, thiols, alcohols, amines,hydrazines, hydrazides, sulphonic acid esters, oxyphosphonium salts) orother chemical moieties which can be utilized in the attachment offurther components. A particularly preferred PEG derivate in thiscontext is alpha-Methoxy-omega-mercapto poly(ethylene glycol). In eachcase, the SH-moiety, e.g. of a cysteine or of any further (modified)amino acid or compound, may be present at the terminal ends orinternally at any position of hydrophilic polymers P¹ and P³. As definedherein, each of hydrophilic polymers P¹ and P³ typically exhibits atleast one —SH-moiety preferably at one terminal end, but may alsocontain two or even more —SH-moieties, which may be used to additionallyattach further components as defined herein, preferably furtherfunctional peptides or proteins e.g. a ligand, an amino acid component(AA) or (AA)_(x), antibodies, cell penetrating peptides or enhancerpeptides (e.g. TAT, KALA), etc.

In the context of the entire formula (V) of the inventive polymericcarrier may be preferably defined as follows:

L-P¹—S-[Cys-P²-Cys]_(n)-S—P³-L   formula (VI)

wherein L, P¹, P², P³ and n are as defined herein, S is sulphur and eachCys provides for one —SH-moiety for the disulfide bond.

The amino acid component (AA) or (AA)_(x) in the polymeric carrier offormula (V or VI), e.g. as defined above for the polymeric carrierformed by disulfide-crosslinked cationic components may also occur as amixed repetitive amino acid component [(AA)_(x)]_(z), wherein the numberof amino acid components (AA) or (AA)_(x) is further defined by integerz. In this context, z may be selected from a range of about 1 to 30,preferably from a range of about 1 to 15, more preferably 1 to 10 or 1to 5 and even more preferably selected from a number selected from 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, or may be selected from arange formed by any two of the afore mentioned values.

According to a specific and particularly preferred alternative, theamino acid component (AA) or (AA)_(x), preferably written asS-(AA)_(x)-S or [S-(AA)_(x)-S] may be used to modify component P²,particularly the content of component S—P²—S in repetitive component[S—P²—S]^(n) of the polymeric carrier of formula (V) above. This may berepresented in the context of the entire polymeric carrier according toformula (VI) e.g. by following formula (VIa):

L-¹-S—{[S—P²—S]_(a)[S-(AA)_(x)-S]_(b)}—S—P³-L,   formula (VIa)

wherein x, S, L, AA, P¹, P² and P³ are preferably as defined herein. Informula (VIa) above, any of the single components [S—P²—S] and[S-(AA)_(x)-S] may occur in any order in the subformula{[S—P²—S]_(a)[S-(AA)_(x)-S]_(b)}. The numbers of single components[S—P²—S] and [S-(AA)_(x)—S] in the subformula{[S—P²—S]_(a)[S-(AA)_(x)-S]_(b)} are determined by integers a and b,wherein a+b=n. n is an integer and is defined as above for formula (V).

According to another embodiment, the polymeric carrier, which may beused to complex e.g. the at least one RNA of the RNA vaccine encoding atleast one antigen or an adjuvant nucleic acid in the inventivevaccine/inhibitor combination or single components thereof, e.g. of theabove mentioned cationic or polycationic peptides, proteins or polymersor further components, e.g. (AA), may be further modified with a ligand,preferably a carbohydrate, more preferably a sugar, even more preferablymannose.

According to one specific embodiment, the entire polymeric carrier maybe formed by a polymerization condensation (of at least one) of theabove mentioned cationic or polycationic peptides, proteins or polymersor further components, e.g. (AA), via their —SH-moieties in a first stepand complexing e.g. the at least one RNA of the RNA vaccine encoding atleast one antigen or an adjuvant nucleic acid in the inventivevaccine/inhibitor combination to such a polymeric carrier in a secondstep. The polymeric carrier may thus contain a number of at least one oreven more of the same or different of the above defined cationic orpolycationic peptides, proteins or polymers or further components, e.g.(AA), the number preferably determined by the above range.

According to one alternative specific embodiment, the polymeric carrier,which may be used to complex e.g. the at least one RNA of the RNAvaccine encoding at least one antigen or an adjuvant nucleic acid in theinventive vaccine/inhibitor combination is formed by carrying out thepolymerization condensation of at least one of the above mentionedcationic or polycationic peptides, proteins or polymers or furthercomponents, e.g. (AA), via their —SH-moieties simultaneously tocomplexing e.g. the at least one RNA of the RNA vaccine encoding atleast one antigen or an adjuvant nucleic acid in the inventivevaccine/inhibitor combination to the (in situ prepared) polymericcarrier. Likewise, the polymeric carrier may thus also here contain anumber of at least one or even more of the same or different of theabove defined cationic or polycationic peptides, proteins or polymers orfurther components, e.g. (AA), the number preferably determined by theabove range.

N/P ratio: The N/P ratio is a measure of the ionic charge of thecationic (side chain) component of the cationic or polycationic compoundor of the polymeric carrier used as carrier or complexation agent asdefined herein. In particular, if the cationic properties of thecationic component are generated by nitrogens (e.g. of the amino acidside chains), the N/P ratio expresses the ratio of basic nitrogen atomsto phosphate residues in the nucleotide backbone, considering that (sidechain) nitrogen atoms in the cationic component of the cationic orpolycationic compound or of the polymeric carrier contribute to positivecharges and phosphate of the phosphate backbone of the nucleic acidcargo e.g. the at least one RNA coding for at least one antigencomprised in the RNA vaccine or an adjuvant nucleic acid contribute tothe negative charge. Generally, one phosphate provides one negativecharge, e.g. one nucleotide in the cargo nucleic acid molecule providesone negative charge. It may be calculated on the basis that, forexample, 1 μg RNA typically contains about 3 nmol phosphate residues,provided that RNA exhibits a statistical distribution of bases.Additionally, 1 nmol peptide typically contains about x nmol nitrogenresidues, dependent on the molecular weight and the number of its(cationic) amino acids.

Zetapotential: The “zetapotential” is a widely used parameter for theelectrical surface charge of a particle. It is typically determined bymoving the charged particle through an electrical field. In the contextof the present invention, the zetapotential is the preferred parameterfor characterizing the charge of a particle, e.g. of a complexcomprising as carrier or complexation agent a cationic or polycationiccompound and/or a polymeric carrier and as nucleic acid cargo the atleast one RNA coding for at least one antigen of the RNA vaccine or anadjuvant nucleic acid. Thus, in the context of the present invention,the charge of a particle is preferably determined by determining thezetapotential by the laser Doppler electrophoresis method using aZetasizer Nano instrument (Malvern Instruments, Malvern, UK) at 25° C.and a scattering angle of 173°. The surface charge of a given particlealso depends on the ionic strength of the utilized matrix (e.g. saltcontaining buffer) and the pH of the solution. Therefore, the actualzetapotential of a given complex at a charge ratio (N/P) may differslightly between different buffers used for injection. For themeasurement, the particles, such as complexes comprising as carrier orcomplexation agent a cationic or polycationic compound and/or apolymeric carrier and as nucleic acid cargo the at least one RNA codingfor at least one antigen of the RNA vaccine or an adjuvant nucleic acidaccording to the invention are preferably suspended in Ringer Lactatesolution. In a specific embodiment the present invention refers to theuse of a negatively charged complex under the conditions of a giveninjection buffer, preferably under the conditions of a Ringer lactatesolution, assessed by its Zetapotential. A Ringer lactate solutionaccording to the present invention preferably contains 130 mmol/L sodiumions, 109 mmol/L chloride ions, 28 mmol/L lactate, 4 mmol/L potassiumions and 1.5 mmol/L calcium ion. The sodium, chloride, potassium andlactate typically come from NaCl (sodium chloride), NaC₃H₅O₃ (sodiumlactate), CaCl₂ (calcium chloride), and KCl (potassium chloride). Theosmolarity of the Ringer lactate solution is 273 mOsm/L and the pH isadjusted to 6.5.

Immunostimulatory composition: In the context of the invention, animmunostimulatory composition may be typically understood to be acomposition containing at least one component which is able to induce animmune response or from which a component which is able to induce animmune response is derivable. Such immune response may be preferably aninnate immune response or a combination of an adaptive and an innateimmune response. Preferably, an immunostimulatory composition in thecontext of the invention contains at least oneimmunostimulating/adjuvant nucleic acid molecule, more preferably anRNA, for example an mRNA molecule. The immunostimulatory component, suchas the mRNA may be complexed with a suitable carrier. Thus, theimmunostimulatory composition may comprise an mRNA/carrier-complex.Furthermore, the immunostimulatory composition may comprise an adjuvantand/or a suitable vehicle for the immunostimulatory component, such asthe mRNA.

Adjuvant nucleic acid: An adjuvant nucleic acid, as used herein, ispreferably selected from nucleic acids which are known to bind to TLRreceptors. Such an adjuvant nucleic acid can be in the form of a(n)(immunostimulatory) CpG nucleic acid, in particular CpG-RNA or CpG-DNA,which preferably induces an innate immune response. A CpG-RNA or CpG-DNAused according to the invention can be a single-stranded CpG-DNA (ssCpG-DNA), a double-stranded CpG-DNA (dsDNA), a single-stranded CpG-RNA(ss CpG-RNA) or a double-stranded CpG-RNA (ds CpG-RNA). The CpG nucleicacid used according to the invention is preferably in the form ofCpG-RNA, more preferably in the form of single-stranded CpG-RNA (ssCpG-RNA). Also preferably, such CpG nucleic acids have a length asdescribed above. Preferably, the CpG motifs are unmethylated.Furthermore, an adjuvant nucleic acid, as used herein, can be animmunostimulatory RNA (isRNA), which preferably elicits an innate immuneresponse.

Preferably, an adjuvant nucleic acid, preferably an immunostimulatoryRNA (isRNA), as used herein, may comprise any nucleic acid sequenceknown to be immunostimulatory, including, e.g., nucleic acid sequencesrepresenting and/or encoding ligands of TLRs, preferably selected fromhuman family members TLR1-TLR10 or murine family members TLR1-TLR13,more preferably selected from (human) family members TLR1-TLR10, evenmore preferably from TLR7 and TLR8, ligands for intracellular receptorsfor RNA (such as RIG-I or MDA-5, etc.) (see e.g. Meylan, E., Tschopp, J.(2006). Toll-like receptors and RNA helicases: two parallel ways totrigger antiviral responses. Mol. Cell 22, 561-569), or any otherimmunostimulatory RNA sequence. Such an adjuvant nucleic acid maycomprise a length of 1000 to 5000, of 500 to 5000, of 5 to 5000, or of 5to 1000, 5 to 500, 5 to 250, of 5 to 100, of 5 to 50 or of 5 to 30nucleotides.

According to a particularly preferred embodiment, an adjuvant nucleicacid sequence, particularly an isRNA, as used herein, may consist of orcomprise a nucleic acid of formula (VII) or (VIII):

G_(l)X_(m)G_(n),   (formula (VII))

wherein:

-   -   G is guanosine, uracil or an analogue of guanosine or uracil;    -   X is guanosine, uracil, adenosine, thymidine, cytosine or an        analogue of the above-mentioned nucleotides;    -   l is an integer from 1 to 40,        -   wherein        -   when 1=1 G is guanosine or an analogue thereof,        -   when 1>1 at least 50% of the nucleotides are guanosine or an            analogue thereof;    -   m is an integer and is at least 3;        -   wherein        -   when m=3 X is uracil or an analogue thereof,        -   when m>3 at least 3 successive uracils or analogues of            uracil occur;    -   n is an integer from 1 to 40,        -   wherein        -   when n=1 G is guanosine or an analogue thereof,        -   when n>1 at least 50% of the nucleotides are guanosine or an            analogue thereof.

C_(l)X_(m)C_(n),   (formula (VIII))

wherein:

-   -   C is cytosine, uracil or an analogue of cytosine or uracil;    -   X is guanosine, uracil, adenosine, thymidine, cytosine or an        analogue of the above-mentioned nucleotides;    -   l is an integer from 1 to 40,        -   wherein        -   when 1=1 C is cytosine or an analogue thereof,        -   when 1>1 at least 50% of the nucleotides are cytosine or an            analogue thereof;    -   m is an integer and is at least 3;        -   wherein        -   when m=3 X is uracil or an analogue thereof,        -   when m>3 at least 3 successive uracils or analogues of            uracil occur;    -   n is an integer from 1 to 40,        -   wherein        -   when n=1 C is cytosine or an analogue thereof,        -   when n>1 at least 50% of the nucleotides are cytosine or an            analogue thereof.

The nucleic acids of formula (VII) or (VIII), which may be used as anadjuvant nucleic acid sequence, particularly an isRNA, may be relativelyshort nucleic acid molecules with a typical length of approximately from5 to 100 (but may also be longer than 100 nucleotides for specificembodiments, e.g. up to 200 nucleotides), from 5 to 90 or from 5 to 80nucleotides, preferably a length of approximately from 5 to 70, morepreferably a length of approximately from 8 to 60 and, more preferably alength of approximately from 15 to 60 nucleotides, more preferably from20 to 60, most preferably from 30 to 60 nucleotides. If the nucleic acidof formula (VII) or (VIII) has a maximum length of e.g. 100 nucleotides,m will typically be<=98. The number of nucleotides G in the nucleic acidof formula (I) is determined by l or n. l and n, independently of oneanother, are each an integer from 1 to 40, wherein when 1 or n=1 G isguanosine or an analogue thereof, and when 1 or n>1 at least 50% of thenucleotides are guanosine or an analogue thereof. For example, withoutimplying any limitation, when l or n=4 G_(l) or G_(n) can be, forexample, a GUGU, GGUU, UGUG, UUGG, GUUG, GGGU, GGUG, GUGG, UGGG or GGGG,etc.; when l or n=5 G_(l) or G_(n) can be, for example, a GGGUU, GGUGU,GUGGU, UGGGU, UGGUG, UGUGG, UUGGG, GUGUG, GGGGU, GGGUG, GGUGG, GUGGG,UGGGG, or GGGGG, etc.; etc. A nucleotide adjacent to X_(m) in thenucleic acid of formula (VII) according to the invention is preferablynot a uracil. Similarly, the number of nucleotides C in the nucleic acidof formula (VIII) according to the invention is determined by l or n. land n, independently of one another, are each an integer from 1 to 40,wherein when 1 or n=1 C is cytosine or an analogue thereof, and when lor n>1 at least 50% of the nucleotides are cytosine or an analoguethereof. For example, without implying any limitation, when l or n=4,C_(l) or C_(n) can be, for example, a CUCU, CCUU, UCUC, UUCC, CUUC,CCCU, CCUC, CUCC, UCCC or CCCC, etc.; when l or n=5 C_(l) or C_(n) canbe, for example, a CCCUU, CCUCU, CUCCU, UCCCU, UCCUC, UCUCC, UUCCC,CUCUC, CCCCU, CCCUC, CCUCC, CUCCC, UCCCC, or CCCCC, etc.; etc. Anucleotide adjacent to X_(m) in the nucleic acid of formula (VIII)according to the invention is preferably not a uracil. Preferably, forformula (VII), when l or n>1, at least 60%, 70%, 80%, 90% or even 100%of the nucleotides are guanosine or an analogue thereof, as definedabove. The remaining nucleotides to 100% (when guanosine constitutesless than 100% of the nucleotides) in the flanking sequences G_(l)and/or G_(n) are uracil or an analogue thereof, as defined hereinbefore.

Also preferably, l and n, independently of one another, are each aninteger from 2 to 30, more preferably an integer from 2 to 20 and yetmore preferably an integer from 2 to 15. The lower limit of l or n canbe varied if necessary and is at least 1, preferably at least 2, morepreferably at least 3, 4, 5, 6, 7, 8, 9 or 10. This definition appliescorrespondingly to formula (VIII).

According to a further particularly preferred embodiment, an adjuvantnucleic acid sequence, particularly an isRNA, as used herein, mayconsist of or comprise a nucleic acid of formula (IX) or (X):

(N_(u)G_(l)X_(m)G_(n)N_(v))_(a),   (formula (IX))

wherein:

-   -   G is guanosine (guanine), uridine (uracil) or an analogue of        guanosine (guanine) or uridine (uracil), preferably guanosine        (guanine) or an analogue thereof;    -   X is guanosine (guanine), uridine (uracil), adenosine (adenine),        thymidine (thymine), cytidine (cytosine), or an analogue of        these nucleotides (nucleosides), preferably uridine (uracil) or        an analogue thereof;    -   N is a nucleic acid sequence having a length of about 4 to 50,        preferably of about 4 to 40, more preferably of about 4 to 30 or        4 to 20 nucleic acids, each N independently being selected from        guanosine (guanine), uridine (uracil), adenosine (adenine),        thymidine (thymine), cytidine (cytosine) or an analogue of these        nucleotides (nucleosides);    -   a is an integer from 1 to 20, preferably from 1 to 15, most        preferably from 1 to 10;    -   l is an integer from 1 to 40,        -   wherein        -   when l=1, G is guanosine (guanine) or an analogue thereof,        -   when l>1, at least 50% of these nucleotides (nucleosides)            are guanosine (guanine) or an analogue thereof;    -   m is an integer and is at least 3;        -   wherein        -   when m=3, X is uridine (uracil) or an analogue thereof, and        -   when m>3, at least 3 successive uridines (uracils) or            analogues of uridine (uracil) occur;    -   n is an integer from 1 to 40,        -   wherein        -   when n=1, G is guanosine (guanine) or an analogue thereof,        -   when n>1, at least 50% of these nucleotides (nucleosides)            are guanosine (guanine) or an analogue thereof;    -   u, v may be independently from each other an integer from 0 to        50, preferably wherein when u=0, v≥1, or        -   when v=0, u≥1;

wherein the nucleic acid molecule of formula (IX) has a length of atleast 50 nucleotides, preferably of at least 100 nucleotides, morepreferably of at least 150 nucleotides, even more preferably of at least200 nucleotides and most preferably of at least 250 nucleotides.

(N_(u)C_(l)X_(m)C_(n)N_(v))_(a)   (formula (X))

wherein:

-   -   C is cytidine (cytosine), uridine (uracil) or an analogue of        cytidine (cytosine) or uridine (uracil), preferably cytidine        (cytosine) or an analogue thereof;    -   X is guanosine (guanine), uridine (uracil), adenosine (adenine),        thymidine (thymine), cytidine (cytosine) or an analogue of the        above-mentioned nucleotides (nucleosides), preferably uridine        (uracil) or an analogue thereof;    -   N is each a nucleic acid sequence having independent from each        other a length of about 4 to 50, preferably of about 4 to 40,        more preferably of about 4 to 30 or 4 to 20 nucleic acids, each        N independently being selected from guanosine (guanine), uridine        (uracil), adenosine (adenine), thymidine (thymine), cytidine        (cytosine) or an analogue of these nucleotides (nucleosides);    -   a is an integer from 1 to 20, preferably from 1 to 15, most        preferably from 1 to 10;    -   l is an integer from 1 to 40,        -   wherein        -   when l=1, C is cytidine (cytosine) or an analogue thereof,        -   when l>1, at least 50% of these nucleotides (nucleosides)            are cytidine (cytosine) or an analogue thereof;    -   m is an integer and is at least 3;        -   wherein        -   when m=3, X is uridine (uracil) or an analogue thereof,        -   when m>3, at least 3 successive uridines (uracils) or            analogues of uridine (uracil) occur;    -   n is an integer from 1 to 40,        -   wherein        -   when n=1, C is cytidine (cytosine) or an analogue thereof,        -   when n>1, at least 50% of these nucleotides (nucleosides)            are cytidine (cytosine) or an analogue thereof.    -   u, v may be independently from each other an integer from 0 to        50, preferably wherein when u=0, v≥1, or        -   when v=0, u≥1;

wherein the nucleic acid molecule of formula (X) according to theinvention has a length of at least 50 nucleotides, preferably of atleast 100 nucleotides, more preferably of at least 150 nucleotides, evenmore preferably of at least 200 nucleotides and most preferably of atleast 250 nucleotides.

Any of the definitions given above in formulae (VII) and (VIII), e.g.for elements N (i.e. N_(u) and N_(v)) and X (X_(m)), particularly thecore structure as defined above, as well as for integers a, l, m, n, uand v, similarly apply to elements of formula (IX) and (X)correspondingly. The definition of bordering elements N_(u) and N_(v) informula (X) is identical to the definitions given above for N_(u) andN_(v) in formula (IX).

Immunostimulatory RNA: An immunostimulatory RNA (isRNA) in the contextof the invention may typically be an RNA that is able to induce aninnate immune response. It usually does not have an open reading frameand thus does not provide a peptide-antigen or immunogen but elicits aninnate immune response e.g. by binding to a specific kind ofToll-like-receptor (TLR) or other suitable receptors. However, of coursealso mRNAs having an open reading frame and coding for a peptide/proteinmay induce an innate immune response and, thus, may be immunostimulatoryRNAs. Preferably, the immunostimulatory RNA may be a single-stranded, adouble-stranded or a partially double-stranded RNA, more preferably asingle-stranded RNA, and/or a circular or linear RNA, more preferably alinear RNA. More preferably, the immunostimulatory RNA may be a (linear)single-stranded RNA. Even more preferably, the immunostimulatory RNA maybe a (long) (linear) (single-stranded) non-coding RNA. In this contextit is particular preferred that the isRNA carries a triphosphate at its5′-end which is the case for in vitro transcribed RNA. Animmunostimulatory RNA may also occur as a short RNA oligonucleotide asdefined herein. An immunostimulatory RNA as used herein may furthermorebe selected from any class of RNA molecules, found in nature or beingprepared synthetically, and which can induce an innate immune responseand may support an adaptive immune response induced by an antigen.Furthermore, (classes of) immunostimulatory RNA molecules, used as afurther compound of the inventive vaccine/inhibitor combination, mayinclude any other RNA capable of eliciting an innate immune response.E.g., such an immunostimulatory RNA may include ribosomal RNA (rRNA),transfer RNA (tRNA), messenger RNA (mRNA), and viral RNA (vRNA). Such animmunostimulatory RNA may comprise a length of 1000 to 5000, of 500 to5000, of 5 to 5000, or of 5 to 1000, 5 to 500, 5 to 250, of 5 to 100, of5 to 50 or of 5 to 30 nucleotides.

siRNA: A small interfering RNA (siRNA), is of interest particularly inconnection with the phenomenon of RNA interference. The in vitrotechnique of RNA interference (RNAi) is based on double-stranded RNAmolecules (dsRNA), which trigger the sequence-specific suppression ofgene expression (Zamore (2001) Nat. Struct. Biol. 9: 746-750; Sharp(2001) Genes Dev. 5:485-490: Hannon (2002) Nature 41: 244-251). In thetransfection of mammalian cells with long dsRNA, the activation ofprotein kinase R and RnaseL brings about unspecific effects, such as,for example, an interferon response (Stark et al. (1998) Annu. Rev.Biochem. 67: 227-264; He and Katze (2002) Viral Immunol. 15: 95-119).These unspecific effects are avoided when shorter, for example 21- to23-mer, so-called siRNA (small interfering RNA), is used, becauseunspecific effects are not triggered by siRNA that is shorter than 30 bp(Elbashir et al. (2001) Nature 411: 494-498).

In the context of the present invention, siRNAs directed against PD-1,PD-L1 or PD-L2 may thus be double-stranded RNAs (dsRNAs) having a lengthof from 17 to 29, preferably from 19 to 25, and preferably is at least90%, more preferably 95% and especially 100% (of the nucleotides of adsRNA) complementary to a section of the naturally occurring mRNAsequence coding for PD-1, PD-L1 or PD-L2 either to a coding or anon-coding section, preferably a coding section. Such a section of thenaturally occurring mRNA sequence may be termed herein a “targetsequence” and may be any section of the naturally occurring mRNA codingfor PD-1, PD-L1 or PD-L2. 90% complementary means that with a length ofa dsRNA described herein of, for example, 20 nucleotides, the dsRNAcontain not more than 2 nucleotides showing no complementarity with thecorresponding section of the target sequence. The sequence of thedouble-stranded siRNA used according to the invention is, however,preferably wholly complementary in its general structure with a sectionof the target sequence. In this context the nucleic acid molecule of thecomplex may be a dsRNA having the general structure 5′-(N₁₇₋₂₉)-3′,preferably having the general structure 5′-(N₁₉₋₂₅)-3′, more preferablyhaving the general structure 5′-(N₁₉₋₂₄)-3′, or yet more preferablyhaving the general structure 5′-(N₂₁₋₂₃)-3′, wherein for each generalstructure each N is a (preferably different) nucleotide of a section ofthe target sequence, preferably being selected from a continuous numberof 17 to 29 nucleotides of a section of the target sequence, and beingpresent in the general structure 5′-(N₁₇₋₂₉)-3′ in their natural order.In principle, all the sections having a length of from 17 to 29,preferably from 19 to 25, base pairs that occur in the target sequencecan serve for preparation of a dsRNA as defined herein. Equally, dsRNAsused as siRNAs can also be directed against mRNA sequences that do notlie in the coding region, in particular in the 5′ non-coding region ofthe target sequence, for example, therefore, against non-coding regionsof the target sequence having a regulatory function. The target sequenceof the dsRNA used as siRNA can therefore lie in the translated anduntranslated region of the target sequence and/or in the region of thecontrol elements of the mRNA sequence. The target sequence for a dsRNAused as siRNA directed against PD-1, PD-L1 or PD-L2 can also lie in theoverlapping region of untranslated and translated sequence; inparticular, the target sequence can comprise at least one nucleotideupstream of the start triplet of the coding region of the mRNA sequence.

Open reading frame: An open reading frame (ORF) in the context of theinvention may typically be a sequence of several nucleotide tripletswhich may be translated into a peptide or protein. An open reading framepreferably contains a start codon, i.e. a combination of threesubsequent nucleotides coding usually for the amino acid methionine (ATGor AUG), at its 5′-end and a subsequent region which usually exhibits alength which is a multiple of 3 nucleotides. An ORF is preferablyterminated by a stop-codon (e.g., TAA, TAG, TGA). Typically, this is theonly stop-codon of the open reading frame. Thus, an open reading framein the context of the present invention is preferably a nucleotidesequence, consisting of a number of nucleotides that may be divided bythree, which starts with a start codon (e.g. ATG or AUG) and whichpreferably terminates with a stop codon (e.g., TAA, TGA, or TAG or UAA,UAG, UGA, respectively). The open reading frame may be isolated or itmay be incorporated in a longer nucleic acid sequence, for example in avector or an mRNA. An open reading frame may also be termed “proteincoding region” or “coding region”.

IRES (internal ribosomal entry site) sequence: An IRES can function as asole ribosome binding site, but it can also serve to provide a bi- oreven multicistronic RNA as defined herein which codes for severalproteins or peptides, which are to be translated by the ribosomesindependently of one another. Examples of IRES sequences which can beused according to the invention are those from picornaviruses (e.g.FMDV), pestiviruses (CFFV), polioviruses (PV), encephalomyocarditisviruses (ECMV), foot and mouth disease viruses (FMDV), hepatitis Cviruses (HCV), classical swine fever viruses (CSFV), mouse leukoma virus(MLV), simian immunodeficiency viruses (SIV) or cricket paralysisviruses (CrPV).

Fragment or part of an (protein/peptide) antigen: Fragments or parts ofa (protein/peptide) antigen in the context of the present invention aretypically understood to be peptides corresponding to a continuous partof the amino acid sequence of a (protein/peptide) antigen, preferablyhaving a length of about 6 to about 20 or even more amino acids, e.g.parts as processed and presented by MHC class I molecules, preferablyhaving a length of about 8 to about 10 amino acids, e.g. 8, 9, or 10,(or even 11, or 12 amino acids), or fragments as processed and presentedby MHC class II molecules, preferably having a length of about 13 ormore amino acids, e.g. 13, 14, 15, 16, 17, 18, 19, 20 or even more aminoacids, wherein these fragments may be selected from any part of theamino acid sequence. These fragments are typically recognized by T cellsin form of a complex consisting of the peptide fragment and an MHCmolecule, i.e. the fragments are typically not recognized in theirnative form. Fragments or parts of the (protein/peptide) antigens asdefined herein may also comprise epitopes or functional sites of those(protein/peptide) antigens. Preferably, fragments or parts of a(protein/peptide) antigen in the context of the invention are orcomprise epitopes, or do have antigenic characteristics, eliciting anadaptive immune response. Therefore, fragments of (protein/peptide)antigens may comprise at least one epitope of those (protein/peptide)antigens. Furthermore, also domains of a (protein/peptide) antigen, likethe extracellular domain, the intracellular domain or the transmembranedomain and shortened or truncated versions of a (protein/peptide)antigen may be understood to comprise a fragment of a (protein/peptide)antigen.

Variants of proteins: “Variants” of proteins or peptides as defined inthe context of the present invention may be generated, having an aminoacid sequence which differs from the original sequence in one or moremutation(s), such as one or more substituted, inserted and/or deletedamino acid(s). Preferably, these variants have the same biologicalfunction or specific activity compared to the full-length nativeprotein, e.g. its specific antigenic property. “Variants” of proteins orpeptides as defined in the context of the present invention may compriseconservative amino acid substitution(s) compared to their native, i.e.non-mutated physiological, sequence. Those amino acid sequences as wellas their encoding nucleotide sequences in particular fall under the termvariants as defined herein. Substitutions in which amino acids, whichoriginate from the same class, are exchanged for one another are calledconservative substitutions. In particular, these are amino acids havingaliphatic side chains, positively or negatively charged side chains,aromatic groups in the side chains or amino acids, the side chains ofwhich can enter into hydrogen bridges, e.g. side chains which have ahydroxyl function. This means that e.g. an amino acid having a polarside chain is replaced by another amino acid having a likewise polarside chain, or, for example, an amino acid characterized by ahydrophobic side chain is substituted by another amino acid having alikewise hydrophobic side chain (e.g. serine (threonine) by threonine(serine) or leucine (isoleucine) by isoleucine (leucine)). Insertionsand substitutions are possible, in particular, at those sequencepositions which cause no modification to the three-dimensional structureor do not affect the binding region. Modifications to athree-dimensional structure by insertion(s) or deletion(s) can easily bedetermined e.g. using CD spectra (circular dichroism spectra) (Urry,1985, Absorption, Circular Dichroism and ORD of Polypeptides, in: ModernPhysical Methods in Biochemistry, Neuberger et al. (ed.), Elsevier,Amsterdam).

Furthermore, variants of proteins or peptides as defined herein, whichmay be encoded by a nucleic acid molecule, may also comprise thosesequences, wherein nucleotides of the nucleic acid are exchangedaccording to the degeneration of the genetic code, without leading to analteration of the respective amino acid sequence of the protein orpeptide, i.e. the amino acid sequence or at least part thereof may notdiffer from the original sequence in one or more mutation(s) within theabove meaning.

In order to determine the percentage to which two sequences areidentical, e.g. nucleic acid sequences or amino acid sequences asdefined herein, preferably the amino acid sequences encoded by a nucleicacid sequence of the polymeric carrier as defined herein or the aminoacid sequences themselves, the sequences can be aligned in order to besubsequently compared to one another. Therefore, e.g. a position of afirst sequence may be compared with the corresponding position of thesecond sequence. If a position in the first sequence is occupied by thesame component (residue) as is the case at a position in the secondsequence, the two sequences are identical at this position. If this isnot the case, the sequences differ at this position. If insertions occurin the second sequence in comparison to the first sequence, gaps can beinserted into the first sequence to allow a further alignment. Ifdeletions occur in the second sequence in comparison to the firstsequence, gaps can be inserted into the second sequence to allow afurther alignment. The percentage to which two sequences are identicalis then a function of the number of identical positions divided by thetotal number of positions including those positions which are onlyoccupied in one sequence. The percentage to which two sequences areidentical can be determined using a mathematical algorithm. A preferred,but not limiting, example of a mathematical algorithm which can be usedis the algorithm of Karlin et al. (1993), PNAS USA, 90:5873-5877 orAltschul et al. (1997), Nucleic Acids Res., 25:3389-3402. Such analgorithm is integrated in the BLAST program. Sequences which areidentical to the sequences of the present invention to a certain extentcan be identified by this program. A “variant” of a protein or peptidemay have at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% amino acididentity over a stretch of 10, 20, 30, 50, 75 or 100 amino acids of suchprotein or peptide, preferably over the full-length sequence thatvariant is derived from. Analogously, a “variant” of a nucleic acidsequence may have at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%nucleotide identity over a stretch of 10, 20, 30, 50, 75 or 100nucleotide of such nucleic acid sequence, preferably over thefull-length sequence the variant is derived from.

Derivative of a protein or peptide: A derivative of a peptide or proteinis typically understood to be a molecule that is derived from anothermolecule, such as said peptide or protein. A “derivative” of a peptideor protein also encompasses fusions comprising a peptide or protein usedin the present invention. For example, the fusion comprises a label,such as, for example, an epitope, e.g., a FLAG epitope or a V5 epitope.For example, the epitope is a FLAG epitope. Such a tag is useful for,for example, purifying the fusion protein.

Pharmaceutically effective amount: A pharmaceutically effective amountin the context of the invention is typically understood to be an amountthat is sufficient to induce a pharmaceutical effect, such as an immuneresponse, altering a pathological level of an expressed peptide orprotein, or substituting a lacking gene product, e.g., in case of apathological situation.

Vehicle: A vehicle is typically understood to be a material that issuitable for storing, transporting, and/or administering a compound,such as a pharmaceutically active compound. For example, it may be aphysiologically acceptable liquid which is suitable for storing,transporting, and/or administering a pharmaceutically active compound.PD-1 pathway: Members of the PD-1 pathway are all proteins which areassociated with PD-1 signaling. On the one hand these might be proteinswhich induce PD-1 signaling upstream of PD-1 as e.g. the ligands of PD-1PD-L1 and PD-L2 and the signal transduction receptor PD-1. On the otherhand these might be signal transduction proteins downstream of PD-1receptor. Particularly preferred as members of the PD-1 pathway in thecontext of the present invention are PD-1, PD-L1 and PD-L2.

PD-1 pathway inhibitor: In the context of the present invention, a PD-1pathway inhibitor is preferably defined herein as a compound capable toimpair the PD-1 pathway signaling, preferably signaling mediated by thePD-1 receptor. Therefore, the PD-1 pathway inhibitor may be anyinhibitor directed against any member of the PD-1 pathway capable ofantagonizing PD-1 pathway signaling. In this context, the inhibitor maybe an antagonistic antibody as defined herein, targeting any member ofthe PD-1 pathway, preferably directed against PD-1 receptor, PD-L1 orPD-L2. This antagonistic antibody may also be encoded by a nucleic acid.Such encoded antibodies are also called “intrabodies” as defined herein.Also, the PD-1 pathway inhibitor may be a fragment of the PD-1 receptoror the PD1-receptor blocking the activity of PD1 ligands. B7-1 orfragments thereof may act as PD1-inhibiting ligands as well.Furthermore, the PD-1 pathway inhibitor may be siRNA (small interferingRNA) or antisense RNA directed against a member of the PD-1 pathway,preferably PD-1, PD-L1 or PD-L2. Additionally, a PD-1 pathway inhibitormay be a protein comprising (or a nucleic acid coding for) an amino acidsequence capable of binding to PD-1 but preventing PD-1 signaling, e.g.by inhibiting PD-1 and B7-H1 or B7-DL interaction. Additionally, a PD-1pathway inhibitor may be a small molecule inhibitor capable ofinhibiting PD-1 pathway signaling, e.g. a PD-1 binding peptide or asmall organic molecule.

Antibody: An antibody may be selected from any antibody, e.g. anyrecombinantly produced or naturally occurring antibodies, known in theart, in particular antibodies suitable for therapeutic, diagnostic orscientific purposes, particularly directed against PD-1, PD-L1 or PD-L2.Herein, the term “antibody” is used in its broadest sense andspecifically covers monoclonal and polyclonal antibodies (including,antagonist, and blocking or neutralizing antibodies) and antibodyspecies with polyepitopic specificity. According to the invention,“antibody” typically comprises any antibody known in the art (e.g. IgM,IgD, IgG, IgA and IgE antibodies), such as naturally occurringantibodies, antibodies generated by immunization in a host organism,antibodies which were isolated and identified from naturally occurringantibodies or antibodies generated by immunization in a host organismand recombinantly produced by biomolecular methods known in the art, aswell as chimeric antibodies, human antibodies, humanized antibodies,bispecific antibodies, intrabodies, i.e. antibodies expressed in cellsand optionally localized in specific cell compartments, and fragmentsand variants of the aforementioned antibodies. In general, an antibodyconsists of a light chain and a heavy chain both having variable andconstant domains. The light chain consists of an N-terminal variabledomain, VL, and a C-terminal constant domain, CL. In contrast, the heavychain of the IgG antibody, for example, is comprised of an N-terminalvariable domain, VH, and three constant domains, CH1 CH2 and CH3. Singlechain antibodies may be used according to the present invention as well.

Antibodies may preferably comprise full-length antibodies, i.e.antibodies composed of the full heavy and full light chains, asdescribed above. However, derivatives of antibodies such as antibodyfragments, variants or adducts may also be used as PD-1 pathwayinhibitor according to the invention. Antibody fragments may be selectedfrom Fab, Fab′, F(ab′)₂, Fc, Facb, pFc′, Fd and Fv fragments of theaforementioned (full-length) antibodies. In general, antibody fragmentsare known in the art. For example, a Fab (“fragment, antigen binding”)fragment is composed of one constant and one variable domain of each ofthe heavy and the light chain. The two variable domains bind the epitopeon specific antigens. The two chains are connected via a disulfidelinkage. A scFv (“single chain variable fragment”) fragment, forexample, typically consists of the variable domains of the light andheavy chains. The domains are linked by an artificial linkage, ingeneral a polypeptide linkage such as a peptide composed of 15-25glycine, proline and/or serine residues.

Polyclonal antibody: Polyclonal antibody typically means mixtures ofantibodies directed to specific antigens or immunogens or epitopes of aprotein which were generated by immunization of a host organism, such asa mammal, e.g. including goat, cattle, swine, dog, cat, donkey, monkey,ape, a rodent such as a mouse, hamster and rabbit. Polyclonal antibodiesare generally not identical, and thus usually recognize differentepitopes or regions from the same antigen. Thus, in such a case,typically a mixture (a composition) of different antibodies will beused, each antibody being directed to specific antigens or immunogens orepitopes of a protein, particularly directed to PD-1, PD-L1 or PD-L2.

Monoclonal antibody: The term “monoclonal antibody” herein typicallyrefers to an antibody obtained from a population of substantiallyhomogeneous antibodies, i.e., the individual antibodies comprising thepopulation are identical except for possible naturally-occurringmutations that may be present in minor amounts. Monoclonal antibodiesare highly specific, being directed to a single antigenic site.Furthermore, in contrast to conventional (polyclonal) antibodypreparations which typically include different antibodies directed todifferent determinants (epitopes), each monoclonal antibody is directedto a single determinant on the antigen. For example, monoclonalantibodies as defined above may be made by the hybridoma method firstdescribed by Kohler and Milstein, Nature, 256:495 (1975), or may be madeby recombinant DNA methods, e.g. as described in U.S. Pat. No.4,816,567. “Monoclonal antibodies” may also be isolated from phagelibraries generated using the techniques described in McCafferty et al.,Nature, 348:552-554 (1990), for example. According to Kohler andMilstein, an immunogen (antigen) of interest is injected into a hostsuch as a mouse and B-cell lymphocytes produced in response to theimmunogen are harvested after a period of time. The B-cells are combinedwith myeloma cells obtained from mouse and introduced into a mediumwhich permits the B-cells to fuse with the myeloma cells, producinghybridomas. These fused cells (hybridomas) are then placed into separatewells of microtiter plates and grown to produce monoclonal antibodies.The monoclonal antibodies are tested to determine which of them aresuitable for detecting the antigen of interest. After being selected,the monoclonal antibodies can be grown in cell cultures or by injectingthe hybridomas into mice. In the context of the present inventionparticularly preferred are monoclonal antibodies directed against PD-1,PD-L1 and PD-L2.

Chimeric antibodies: Chimeric antibodies, which may be used as PD-1pathway inhibitor according to the invention are preferably antibodiesin which the constant domains of an antibody described above arereplaced by sequences of antibodies from other organisms, preferablyhuman sequences.

Humanized antibodies: Humanized (non-human) antibodies, which may beused as PD-1 pathway inhibitor according to the invention are antibodiesin which the constant and variable domains (except for the hypervariabledomains) of an antibody are replaced by human sequences.

Human antibodies: Human antibodies can be isolated from human tissues orfrom immunized non-human host organisms which are transgene for thehuman IgG gene locus. Additionally, human antibodies can be provided bythe use of a phage display.

Bispecific antibodies: Bispecific antibodies in context of the inventionare preferably antibodies which act as an adaptor between an effectorand a respective target by two different F_(a/b)-domains, e.g. for thepurposes of recruiting effector molecules such as toxins, drugs,cytokines etc., targeting effector cells such as CTL, NK cells,makrophages, granulocytes, etc. (see for review: Kontermann R. E., ActaPharmacol. Sin, 2005, 26(1): 1-9). Bispecific antibodies as describedherein are, in general, configured to recognize by two differentF_(a/b)-domains, e.g. two different antigens, immunogens, epitopes,drugs, cells (or receptors on cells), or other molecules (or structures)as described above. Bispecificity means herewith that theantigen-binding regions of the antibodies are specific for two differentepitopes. Thus, different antigens, immunogens or epitopes, etc. can bebrought close together, what, optionally, allows a direct interaction ofthe two components. For example, different cells such as effector cellsand target cells can be connected via a bispecific antibody.Encompassed, but not limited, by the present invention are antibodies orfragments thereof which bind, on the one hand, a soluble antigen and, onthe other hand, an antigen or receptor e.g. PD-1 or its ligands PD-L1and PD-L2 on the surface of a cell, e.g. a tumor cell.

Intrabodies: Intrabodies may be antibodies as defined above. Theseantibodies are intracellular expressed antibodies, and therefore theseantibodies may be encoded by nucleic acids to be used for expression ofthe encoded antibodies. Therefore nucleic acids coding for an antibody,preferably as defined above, particularly an antibody directed against amember of the PD-1 pathway, e.g. PD-1, PD-L1 or PD-L2 may be used asPD-1 pathway inhibitor according to the present invention.

According to a first aspect, the object underlying the present inventionis solved by a vaccine/inhibitor combination comprising:

-   -   (i) as vaccine an RNA vaccine comprising at least one RNA        comprising at least one open reading frame (ORF) coding for at        least one antigen, and    -   (ii) as an inhibitor a composition comprising at least one PD-1        pathway inhibitor.

In the context of the present invention, the term “vaccine/inhibitorcombination” preferably means a combined occurrence of an RNA vaccinecomprising at least one RNA comprising at least one open reading frame(ORF) coding for at least one antigen and of a composition comprising atleast one PD-1 pathway inhibitor. Therefore, this vaccine/inhibitorcombination may occur either as one composition, comprising all thesecomponents in one and the same mixture (e.g. in a pharmaceuticalcomposition), or may occur as a kit of parts, wherein the differentcomponents form different parts of such a kit of parts. This inventivevaccine/inhibitor combination preferably allows to elicit an adaptiveimmune response (and optional an innate immune response) in a patient tobe treated, preferably a mammal, by using as a first component an RNAvaccine, comprising at least one RNA comprising at least one openreading frame encoding at least one antigen, preferably encoding atumour antigen or a pathogenic antigen. The inhibitor of the inventivevaccine/inhibitor combination, preferably a PD-1 pathway inhibitor mayantagonize PD-1 pathway signaling by preferably inhibiting orsuppressing signal transduction mediated by the PD-1 receptor. Thus, theadministration of the vaccine and the inhibitor may occur eithersimultaneously or timely staggered, either at the same site ofadministration or at different sites of administration, as furtheroutlined below. Such a vaccine/inhibitor combination may induce anactive immune response and thereby prevents e.g. tumour growth orinduces tumour regression. The inventive vaccine/inhibitor combinationis thus suitable to effectively stimulate antigen-specific immuneresponses against cancer and pathogen infected cells. More precisely,the inventive vaccine/inhibitor combination is particularly suitable inthe treatment of tumour diseases and infectious diseases which may beassociated with an overexpression of PD-1, PD-L1 or PD-L2 and to furtherimprove the immune response against such tumour cells and infectedcells.

The invention is therefore based on the surprising finding that thecombination of an RNA vaccine and a PD-1 pathway inhibitor shows anextremely advantageous inhibition of tumour growth resulting in enhancedsurvival which could not be expected from the prior art. Thus, thecombined treatment with an RNA vaccine, e.g. coding for a specificantigen (active vaccination) such as a tumour antigen, and with aninhibitor directed to a member of the PD-1 pathway, particularly PD-1receptor or its ligands PD-L1 and PD-L2, could strongly decrease theharmful impact of a disease to be treated, e.g. the growth rate of atumour. In this context, the inventors surprisingly found that treatmentwith an RNA vaccine comprising an RNA coding for a tumor antigen incombination with a PD-1 pathway inhibitor unexpectedly inhibited tumorgrowth resulting in an improved survival of tumor challenged mice in asynergistic manner as evidenced by the occurrence of 50% completeresponses.

As a first component, the inventive vaccine/inhibitor combinationincludes as a vaccine an RNA vaccine which comprises at least one RNAcomprising at least one open reading frame (ORF) coding for at least oneantigen, preferably a tumour antigen or a pathogenic antigen.

According to the invention, the RNA vaccine of the inventivevaccine/inhibitor combination preferably comprises at least one RNAcomprising at least one open reading frame encoding at least one antigenas defined herein.

The at least one RNA of the RNA vaccine may be selected from any RNAsuitable to encode an amino acid sequence, preferably from a messengerRNA (mRNA).

However other forms of RNA may likewise find its application in carryingout the teaching of the present invention. For example, the RNA may be avirus derived RNA such as RNA of a retrovirus or an RNA replicon asdefined herein e.g. derived from an alphavirus.

In specific embodiments the at least one RNA of the RNA vaccine does notcomprise or does not consist of a lentiviral vector or aadenoviral/adeno-associated vector.

In a specific embodiment, the RNA vaccine comprises or consists ofisolated RNA as defined herein.

Furthermore, the at least one RNA of the RNA vaccine of the inventivevaccine/inhibitor combination may be a single- or a double-stranded RNA(which may also be regarded as an RNA (molecule) due to non-covalentassociation of two single-stranded RNA (molecules)) or a partiallydouble-stranded or partially single stranded RNA, which are at leastpartially self-complementary (both of these partially double-stranded orpartially single stranded RNA molecules are typically formed by a longerand a shorter single-stranded RNA molecule or by two single strandedRNA-molecules, which are about equal in length, wherein onesingle-stranded RNA molecule is in part complementary to the othersingle-stranded RNA molecule and both thus form a double-stranded RNAmolecule in this region, i.e. a partially double-stranded or partiallysingle stranded RNA). Preferably, the at least one RNA of the RNAvaccine of the inventive vaccine/inhibitor combination may be asingle-stranded RNA. Furthermore, the at least one RNA of the RNAvaccine of the inventive vaccine/inhibitor combination may be a circularor linear RNA, preferably a linear RNA. More preferably, the at leastone RNA of the RNA vaccine of the inventive vaccine/inhibitorcombination may be a (linear) single-stranded RNA.

Preferably, the at least one RNA of the RNA vaccine of the inventivevaccine/inhibitor combination comprises a length of about 5 to about20000, or 100 to about 20000 nucleotides, preferably of about 250 toabout 20000 nucleotides, more preferably of about 500 to about 10000,even more preferably of about 500 to about 5000.

In a particular preferred embodiment of the first aspect of theinvention, the at least one RNA of the RNA vaccine comprising at leastone open reading frame codes for at least one tumour antigen. In thiscontext tumour antigens are preferably located on the surface of the(tumour) cell. Tumour antigens may also be selected from proteins, whichare overexpressed in tumour cells compared to a normal cell (e.g.non-tumor cells).Furthermore, tumour antigens also include antigensexpressed in cells which are (were) not themselves (or originally notthemselves) degenerated but are associated with the supposed tumour.Antigens which are connected with tumour-supplying vessels or(re)formation thereof, in particular those antigens which are associatedwith neovascularization, e.g. growth factors, such as VEGF, bFGF etc.,are also included herein. Antigens associated with a tumour furthermoreinclude antigens from cells or tissues, typically embedding the tumour.Further, some substances (usually proteins or peptides) are expressed inpatients suffering (knowingly or not-knowingly) from a cancer diseaseand they occur in increased concentrations in the body fluids of saidpatients. These substances are also referred to as “tumour antigens”,however they are not antigens in the stringent meaning of an immuneresponse inducing substance. The class of tumour antigens can be dividedfurther into tumour-specific antigens (TSAs) andtumour-associated-antigens (TAAs). TSAs can only be presented by tumourcells and never by normal “healthy” cells. They typically result from atumour specific mutation. TAAs, which are more common, are usuallypresented by both tumour and healthy cells. These antigens arerecognized and the antigen-presenting cell can be destroyed by cytotoxicT cells. Additionally, tumour antigens can also occur on the surface ofthe tumour in the form of, e.g., a mutated receptor. In this case, theycan be recognized by antibodies.

Further, tumour associated antigens may be classified as tissue-specificantigens, also called melanocyte-specific antigens, cancer-testisantigens and tumour-specific antigens. Cancer-testis antigens aretypically understood to be peptides or proteins of germ-line associatedgenes which may be activated in a wide variety of tumours. Humancancer-testis antigens may be further subdivided into antigens which areencoded on the X chromosome, so-called CT-X antigens, and those antigenswhich are not encoded on the X chromosome, the so-called (non-X CTantigens). Cancer-testis antigens which are encoded on the X-chromosomecomprise, for example, the family of melanoma antigen genes, theso-called MAGE-family. The genes of the MAGE-family may be characterisedby a shared MAGE homology domain (MHD). Each of these antigens, i.e.melanocyte-specific antigens, cancer-testis antigens and tumour-specificantigens, may elicit autologous cellular and humoral immune responses.Accordingly, the tumour antigen encoded by the RNA comprised in the RNAvaccine used in the present invention is preferably amelanocyte-specific antigen, a cancer-testis antigen or atumour-specific antigen, preferably it may be a CT-X antigen, a non-XCT-antigen, a binding partner for a CT-X antigen or a binding partnerfor a non-X CT-antigen or a tumour-specific antigen, more preferably aCT-X antigen, a binding partner for a non-X CT-antigen or atumour-specific antigen.

Particular preferred tumour antigens according to the present inventionare selected from the list consisting of 5T4, 707-AP, 9D7, AFP, AlbZIPHPG1, alpha-5-beta-1-integrin, alpha-5-beta-6-integrin,alpha-actinin-4/m, alpha-methylacyl-coenzyme A racemase, ART-4, ARTC1/m,B7H4, BAGE-1, BCL-2, bcr/abl, beta-catenin/m, BING-4, BRCA1/m, BRCA2/m,CA 15-3/CA 27-29, CA 19-9, CA72-4, CA125, calreticulin, CAMEL, CASP-8/m,cathepsin B, cathepsin L, CD19, CD20, CD22, CD25, CDE30, CD33, CD4,CD52, CD55, CD56, CD80, CDC27/m, CDK4/m, CDKN2A/m, CEA, CLCA2, CML28,CML66, COA-1/m, coactosin-like protein, collage XXIII, COX-2, CT-9/BRD6,Cten, cyclin B1, cyclin D1, cyp-B, CYPB1, DAM-10, DAM-6, DEK-CAN,EFTUD2/m, EGFR, ELF2/m, EMMPRIN, EpCam, EphA2, EphA3, ErbB3, ETV6-AML1,EZH2, FGF-5, FN, Frau-1, G250, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5,GAGE-6, GAGE7b, GAGE-8, GDEP, GnT-V, gp100, GPC3, GPNMB/m, HAGE, HAST-2,hepsin, Her2/neu, HERV-K-MEL, HLA-A*0201-R17I, HLA-A11/m, HLA-A2/m, HNE,homeobox NKX3.1, HOM-TES-14/SCP-1, HOM-TES-85, HPV-E6, HPV-E7, HSP70-2M,HST-2, hTERT, iCE, IGF-1R, IL-13Ra2, IL-2R, IL-5, immature lamininreceptor, kallikrein-2, kallikrein-4, Ki67, KIAA0205, KIAA0205/m,KK-LC-1, K-Ras/m, LAGE-A1, LDLR-FUT, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4,MAGE-A6, MAGE-A9, MAGE-A10, MAGE-Al2, MAGE-B1, MAGE-B2, MAGE-B3,MAGE-B4, MAGE-B5, MAGE-B6, MAGE-B10, MAGE-B16, MAGE-B17, MAGE-C1,MAGE-C2, MAGE-C3, MAGE-D1, MAGE-D2, MAGE-D4, MAGE-E1, MAGE-E2, MAGE-F1,MAGE-H1, MAGEL2, mammaglobin A, MART-1/melan-A, MART-2, MART-2/m, matrixprotein 22, MC1R, M-CSF, ME1/m, mesothelin, MG50/PXDN, MMP11, MN/CAIX-antigen, MRP-3, MUC-1, MUC-2, MUM-1/m, MUM-2/m, MUM-3/m, myosin classI/m, NA88-A, N-acetylglucosaminyltransferase-V, Neo-PAP, Neo-PAP/m,NFYC/m, NGEP, NMP22, NPM/ALK, N-Ras/m, NSE, NY-ESO-B, NY-ESO-1, OA1,OFA-iLRP, OGT, OGT/m, OS-9, OS-9/m, osteocalcin, osteopontin, p15, p190minor bcr-abl, p53, p53/m, PAGE-4, PAI-1, PAI-2, PAP, PART-1, PATE,PDEF, Pim-1-Kinase, Pin-1, Pml/PARalpha, POTE, PRAME, PRDX5/m, prostein,proteinase-3, PSA, PSCA, PSGR, PSM, PSMA, PTPRK/m, RAGE-1, RBAF600/m,RHAMM/CD168, RU1, RU2, S-100, SAGE, SART-1, SART-2, SART-3, SCC,SIRT2/m, Sp17, SSX-1, SSX-2/HOM-MEL-40, SSX-4, STAMP-1, STEAP-1,survivin, survivin-2B, SYT-SSX-1, SYT-SSX-2, TA-90, TAG-72, TARP,TEL-AML1, TGFbeta, TGFbetaRII, TGM-4, TPI/m, TRAG-3, TRG, TRP-1,TRP-2/6b, TRP/INT2, TRP-p8, tyrosinase, UPA, VEGFR1, VEGFR-2/FLK-1, andWT1. Such tumour antigens preferably may be selected from the groupconsisting of p53, CA125, EGFR, Her2/neu, hTERT, PAP, MAGE-A1, MAGE-A3,Mesothelin, MUC-1, GP100, MART-1, Tyrosinase, PSA, PSCA, PSMA, STEAP-1,VEGF, VEGFR1, VEGFR2, Ras, CEA or WT1, and more preferably from PAP,MAGE-A3, WT1, and MUC-1. Such tumour antigens preferably may be selectedfrom the group consisting of MAGE-A1 (e.g. MAGE-A1 according toaccession number M77481), MAGE-A2, MAGE-A3, MAGE-A6 (e.g. MAGE-A6according to accession number NM_005363), MAGE-C1, MAGE-C2, melan-A(e.g. melan-A according to accession number NM_005511), GP100 (e.g.GP100 according to accession number M77348), tyrosinase (e.g. tyrosinaseaccording to accession number NM_000372), surviving (e.g. survivinaccording to accession number AF077350), CEA (e.g. CEA according toaccession number NM_004363), Her-2/neu (e.g. Her-2/neu according toaccession number M11730), WT1 (e.g. WT1 according to accession numberNM_000378), PRAME (e.g. PRAME according to accession number NM_006115),EGFRI (epidermal growth factor receptor 1) (e.g. EGFRI (epidermal growthfactor receptor 1) according to accession number AF288738), MUC1,mucin-1 (e.g. mucin-1 according to accession number NM_002456), SEC61G(e.g. SEC61G according to accession number NM_014302), hTERT (e.g. hTERTaccession number NM_198253), 5T4 (e.g. 5T4 according to accession numberNM_006670), TRP-2 (e.g. TRP-2 according to accession number NM_001922),STEAP1 (Six-transmembrane epithelial antigen of prostate 1), PSCA, PSA,PSMA, etc. In this context it is particularly preferred that the atleast one RNA of the RNA vaccine of the inventive vaccine/inhibitorcombination encodes the tumour antigens selected from PCA,

PSA, PSMA, STEAP and optional MUC-1, or fragments, variants orderivatives thereof.

In a further particularly preferred embodiment the RNA vaccine of theinventive vaccine/inhibitor combination comprises at least one RNAcoding for the tumour antigens selected from NY-ESO-1, MAGE-C1, MAGE-C2,Survivin, optional 5T4 and optional MUC-1, or fragments, variants orderivatives thereof.

Furthermore, tumour antigens also may encompass idiotypic antigensassociated with a cancer or tumour disease, particularly lymphoma or alymphoma associated disease, wherein said idiotypic antigen is animmunoglobulin idiotype of a lymphoid blood cell or a T cell receptoridiotype of a lymphoid blood cell.

In a further particularly preferred embodiment of the first aspect ofthe invention, the at least one RNA of the RNA vaccine comprises atleast one open reading frame coding for at least one pathogenic antigen.Pathogenic antigens are peptide or protein antigens derived from apathogen associated with infectious disease which are preferablyselected from antigens derived from the pathogens Acinetobacterbaumannii, Anaplasma genus, Anaplasma phagocytophilum, Ancylostomabraziliense, Ancylostoma duodenale, Arcanobacterium haemolyticum,Ascaris lumbricoides, Aspergillus genus, Astroviridae, Babesia genus,Bacillus anthracis, Bacillus cereus, Bartonella henselae, BK virus,Blastocystis hominis, Blastomyces dermatitidis, Bordetella pertussis,Borrelia burgdorferi, Borrelia genus, Borrelia spp, Brucella genus,Brugia malayi, Bunyaviridae family, Burkholderia cepacia and otherBurkholderia species, Burkholderia mallei, Burkholderia pseudomallei,Caliciviridae family, Campylobacter genus, Candida albicans, Candidaspp, Chlamydia trachomatis, Chlamydophila pneumoniae, Chlamydophilapsittaci, CJD prion, Clonorchis sinensis, Clostridium botulinum,Clostridium difficile, Clostridium perfringens, Clostridium perfringens,Clostridium spp, Clostridium tetani, Coccidioides spp, coronaviruses,Corynebacterium diphtheriae, Coxiella burnetii, Crimean-Congohemorrhagic fever virus, Cryptococcus neoformans, Cryptosporidium genus,Cytomegalovirus (CMV), Dengue viruses (DEN-1, DEN-2, DEN-3 and DEN-4),Dientamoeba fragilis, Ebolavirus (EBOV), Echinococcus genus, Ehrlichiachaffeensis, Ehrlichia ewingii, Ehrlichia genus, Entamoeba histolytica,Enterococcus genus, Enterovirus genus, Enteroviruses, mainly Coxsackie Avirus and Enterovirus 71 (EV71), Epidermophyton spp, Epstein-Barr Virus(EBV), Escherichia coli 0157:H7, 0111 and 0104:H4, Fasciola hepatica andFasciola gigantica, FFI prion, Filarioidea superfamily, Flaviviruses,Francisella tularensis, Fusobacterium genus, Geotrichum candidum,Giardia intestinalis, Gnathostoma spp, GSS prion, Guanarito virus,Haemophilus ducreyi, Haemophilus influenzae, Helicobacter pylori,Henipavirus (Hendra virus Nipah virus), Hepatitis A Virus, Hepatitis BVirus (HBV), Hepatitis C Virus (HCV), Hepatitis D Virus, Hepatitis EVirus, Herpes simplex virus 1 and 2 (HSV-1 and HSV-2), Histoplasmacapsulatum, HIV (Human immunodeficiency virus), Hortaea werneckii, Humanbocavirus (HBoV), Human herpesvirus 6 (HHV-6) and Human herpesvirus 7(HHV-7), Human metapneumovirus (hMPV), Human papillomavirus (HPV), Humanparainfluenza viruses (HPIV), Japanese encephalitis virus, JC virus,Junin virus, Kingella kingae, Klebsiella granulomatis, Kuru prion, Lassavirus, Legionella pneumophila, Leishmania genus, Leptospira genus,Listeria monocytogenes, Lymphocytic choriomeningitis virus (LCMV),Machupo virus, Malassezia spp, Marburg virus, Measles virus, Metagonimusyokagawai, Microsporidia phylum, Molluscum contagiosum virus (MCV),Mumps virus, Mycobacterium leprae and Mycobacterium lepromatosis,Mycobacterium tuberculosis, Mycobacterium ulcerans, Mycoplasmapneumoniae, Naegleria fowleri, Necator americanus, Neisseriagonorrhoeae, Neisseria meningitidis, Nocardia asteroides, Nocardia spp,Onchocerca volvulus, Orientia tsutsugamushi, Orthomyxoviridae family(Influenza), Paracoccidioides brasiliensis, Paragonimus spp, Paragonimuswestermani, Parvovirus B19, Pasteurella genus, Plasmodium genus,Pneumocystis jirovecii, Poliovirus, Rabies virus, Respiratory syncytialvirus (RSV), Rhinovirus, rhinoviruses, Rickettsia akari, Rickettsiagenus, Rickettsia prowazekii, Rickettsia rickettsii, Rickettsia typhi,Rift Valley fever virus, Rotavirus, Rubella virus, Sabia virus,Salmonella genus, Sarcoptes scabiei, SARS coronavirus, Schistosomagenus, Shigella genus, Sin Nombre virus, Hantavirus, Sporothrixschenckii, Staphylococcus genus, Staphylococcus genus, Streptococcusagalactiae, Streptococcus pneumoniae, Streptococcus pyogenes,Strongyloides stercoralis, Taenia genus, Taenia solium, Tick-borneencephalitis virus (TBEV), Toxocara canis or Toxocara cati, Toxoplasmagondii, Treponema pallidum, Trichinella spiralis, Trichomonas vaginalis,Trichophyton spp, Trichuris trichiura, Trypanosoma brucei, Trypanosomacruzi, Ureaplasma urealyticum, Varicella zoster virus (VZV), Varicellazoster virus (VZV), Variola major or Variola minor, vCJD prion,Venezuelan equine encephalitis virus, Vibrio cholerae, West Nile virus,Western equine encephalitis virus, Wuchereria bancrofti, Yellow fevervirus, Yersinia enterocolitica, Yersinia pestis, and Yersiniapseudotuberculosis.

In this context, particularly preferred are antigens from the pathogensselected from Influenza virus, respiratory syncytial virus (RSV), Herpessimplex virus (HSV), human Papilloma virus (HPV), Human immunodeficiencyvirus (HIV), Plasmodium, Staphylococcus aureus, Dengue virus, Chlamydiatrachomatis, Cytomegalovirus (CMV), Hepatitis B virus (HBV),Mycobacterium tuberculosis, Rabies virus, and Yellow Fever Virus.

The at least one RNA of the RNA vaccine comprising at least one openreading frame coding for at least one antigen according to the firstaspect of the present invention may occur as a mono-, di-, or evenmulticistronic RNA, i.e. an RNA which contains the open reading frame ofone, two or more proteins or peptides. Such open reading frames in di-,or even multicistronic RNAs may be separated by at least one internalribosome entry site (IRES) sequence, e.g. as described herein or bysignal peptides which induce the cleavage of the resulting polypeptidewhich comprises several proteins or peptides.

The at least one RNA of the RNA vaccine of the inventivevaccine/inhibitor combination may be stabilized in order to preventinstability and (fast) degradation of the RNA by various approaches.This instability of RNA is typically due to RNA-degrading enzymes,“RNases” (ribonucleases), wherein contamination with such ribonucleasesmay sometimes completely degrade RNA in solution. Accordingly, thenatural degradation of RNA in the cytoplasm of cells is very finelyregulated and RNase contaminations may be generally removed by specialtreatment prior to use of said compositions, in particular with diethylpyrocarbonate (DEPC). A number of mechanisms of natural degradation areknown in this context in the prior art, which may be utilized as well.E.g., the terminal structure is typically of critical importanceparticularly for an mRNA. As an example, at the 5′ end of naturallyoccurring mRNAs there is usually a so-called cap structure, which is amodified guanosine nucleotide also called 5 ‘Cap structure, and at the3’ end is typically a sequence of up to 200 adenosine nucleotides (theso-called poly-A tail). In a further embodiment, the at least one RNA ofthe RNA vaccine of the inventive vaccine/inhibitor combination comprisesat least one of the following structural elements: a 5′ and/or 3′-UTRsequence, preferably a 5′ and/or 3′-UTR modification, a 5 ‘Capstructure, a poly(C) sequence, a poly-A tail and/or a polyadenylationsignal, preferably as defined herein.

In a further embodiment, the at least one RNA of the RNA vaccine of theinventive vaccine/inhibitor combination preferably comprises at leasttwo of the following structural elements: a 5’ and/or 3′-UTR sequence,preferably a 5′ and/or 3′-UTR modification (e.g. the mutated sequence ofthe 3′-UTR of the (alpha)globin gene (muag)); a histone-stem-loopstructure, preferably a histone-stem-loop in its 3′ untranslated region;a 5′-Cap structure; a poly(C) sequence; a poly-A tail; or apolyadenylation signal, e.g. given a 5′-Cap structure and ahistone-stem-loop and, potentially a poly-A-tail.

In this context it is particularly preferred that the at least one RNAof the RNA vaccine encoding at least one antigen comprised in theinventive vaccine/inhibitor combination has the following structure in5′ to 3′-direction:

a) an optional 5′-UTR sequence comprising a UTR modification

-   -   b) an open reading frame encoding an antigen as defined above;    -   c) a 3′-UTR sequence comprising a UTR modification    -   d) at least one histone stem-loop, optionally without a histone        downstream element 3′ to the histone stem-loop    -   e) a poly(A) sequence or optional a polyadenylation signal; and    -   f) a poly(C) sequence.

In another particular preferred embodiment the at least one RNA of theRNA vaccine encoding at least one antigen in the inventivevaccine/inhibitor combination has the following structure in 5′ to3′-direction:

-   -   a) an optional 5′-UTR sequence comprising a UTR modification    -   b) an open reading frame encoding an antigen as defined above;    -   c) a 3′-UTR sequence comprising a UTR modification    -   d) a poly(A) sequence    -   e) a poly(C) sequence; and    -   f) at least one histone stem-loop.

For further improvement of the resistance to e.g. in vivo degradation(e.g. by an exo- or endo-nuclease), the at least one RNA of the RNAvaccine encoding at least one antigen in the inventive vaccine/inhibitorcombination may be provided as a stabilized nucleic acid, e.g. in theform of a modified nucleic acid as defined herein. According to afurther embodiment of the invention, it is therefore preferred that theat least one RNA of the RNA vaccine encoding at least one antigen in theinventive vaccine/inhibitor combination is stabilized, preferably bybackbone modifications, sugar modifications and/or base modifications,more preferred stabilized by modification of the G/C-content as definedherein. All of these modifications may be introduced into the at leastone RNA without impairing the RNA's function to be translated into theantigen, to be reverse transcribed or to be replicated.

According to another embodiment, the at least one RNA of the RNA vaccineencoding at least one antigen in the inventive vaccine/inhibitorcombination may be modified and thus stabilized by modifying the G(guanosine)/C (cytosine) content of the mRNA, preferably of the openreading frame thereof.

Therein, the G/C content of the at least one RNA of the RNA vaccineencoding at least one antigen in the inventive vaccine/inhibitorcombination is particularly increased compared to the G/C content of theopen reading frame of its particular wild type open reading frame, i.e.the unmodified RNA as defined herein. However, the encoded amino acidsequence of the open reading frame of the at least one RNA of the RNAvaccine encoding at least one antigen in the inventive vaccine/inhibitorcombination RNA is preferably not modified compared to the encoded aminoacid sequence of the particular wild type open reading frame.

According to a further preferred embodiment of the invention, the atleast one RNA of the RNA vaccine encoding at least one antigen in theinventive vaccine/inhibitor combination is optimized for translation(codon-optimized) as defined herein, preferably optimized fortranslation by replacing codons for less frequent tRNAs of a given aminoacid by codons for more frequently occurring tRNAs of the respectiveamino acid.

In this context, it is particularly preferred to link the sequential G/Ccontent which is increased, in particular maximized, in the at least oneRNA of the RNA vaccine encoding at least one antigen in the inventivevaccine/inhibitor combination, with the “frequent” codons withoutmodifying the amino acid sequence of the protein encoded by the openreading frame comprised in the at least one RNA of the RNA vaccine.

In the context of the present invention, the at least one RNA of the RNAvaccine encoding at least one antigen in the inventive vaccine/inhibitorcombination, may be prepared using any method known in the art,including synthetic methods such as e.g. solid phase synthesis, as wellas in vivo propagation like e.g. production of virus-like particles orreplicon particles in cells or in vitro methods, such as in vitrotranscription reactions. Replicons such as self-amplifying RNA based onan alphavirus genome can be produced by constructing DNA plasmidsencoding the self-amplifying RNA using standard molecular techniques.Linearized DNA is transcribed in vitro by, for example, T7 RNApolymerase and the resulting RNA is introduced into cells, e.g. byelectroporation. Replicon particle production can be evaluated inpackaging assays in which in vitro transcribed replicon and defectivehelper RNA are cotransfected into cells (Perri et al., 2003. J. Virol.77(19):10394-403; 12970424).

In a further embodiment the RNA vaccine of the inventivevaccine/inhibitor combination comprises a plurality or more than one,preferably 2 to 10, more preferably 2 to 5, most preferably 2 to 4 ofRNA molecules as defined herein. These RNA vaccines comprise more thanone RNA molecules, preferably encoding different peptides or proteinswhich comprise preferably different tumour antigens or pathogenicantigens.

In this context it is particularly preferred that the RNA vaccine of theinventive vaccine/inhibitor combination comprising a plurality (whichmeans typically more than 1, 2, 3, 4, 5, 6 or more than 10 nucleicacids, e.g. 2 to 10, preferably 2 to 5 nucleic acids) of RNA molecules,particularly for use in the treatment of prostate cancer (PCa) comprisesat least:

-   -   a) an RNA molecule encoding at least one peptide or protein,        wherein said encoded peptide or protein comprises the tumour        antigen PSA, or a fragment, variant or derivative thereof; and    -   b) an RNA molecule encoding at least one peptide or protein,        wherein said encoded peptide or protein comprises the tumour        antigen PSMA, or a fragment, variant or derivative thereof; and    -   c) an RNA molecule encoding at least one peptide or protein,        wherein said encoded peptide or protein comprises the tumour        antigen PSCA, or a fragment, variant or derivative thereof;

d) an RNA molecule encoding at least one peptide or protein, whereinsaid encoded peptide or protein comprises the tumour antigen STEAP-1, ora fragment, variant or derivative thereof; and optional

-   -   e) an RNA molecule encoding at least one peptide or protein,        wherein said encoded peptide or protein comprises the tumour        antigen MUC-1, or a fragment, variant or derivative thereof.

In a further preferred embodiment the RNA vaccine of the inventivevaccine/inhibitor combination comprising a plurality (which meanstypically more than 1, 2, 3, 4, 5, 6 or more than 10 nucleic acids, e.g.2 to 10, preferably 2 to 5 nucleic acids) of RNA molecules, particularlyfor use in the treatment of non-small lung cancer (NSCLC) comprises atleast:

-   -   a) an RNA molecule encoding at least one peptide or protein,        wherein said encoded peptide or protein comprises the tumour        antigen NY-ESO-1, or a fragment, variant or derivative thereof;        and    -   d) an RNA molecule encoding at least one peptide or protein,        wherein said encoded peptide or protein comprises the tumour        antigen MAGE-C1, or a fragment, variant or derivative thereof;        and    -   e) an RNA molecule encoding at least one peptide or protein,        wherein said encoded peptide or protein comprises the tumour        antigen MAGE-C2, or a fragment, variant or derivative thereof;    -   f) an RNA molecule encoding at least one peptide or protein,        wherein said encoded peptide or protein comprises the tumour        antigen Survivin, or a fragment, variant or derivative thereof;        and optional    -   g) an RNA molecule encoding at least one peptide or protein,        wherein said encoded peptide or protein comprises the tumour        antigen 5T4, or a fragment, variant or derivative thereof; and        optional    -   h) an RNA molecule encoding at least one peptide or protein,        wherein said encoded peptide or protein comprises the tumour        antigen MUC-1, or a fragment, variant or derivative thereof.

According to one embodiment of the present invention, the at least oneRNA of the RNA vaccine encoding at least one antigen in the inventivevaccine/inhibitor combination may be administered naked without beingassociated with any further vehicle, transfection or complexation agentfor increasing the transfection efficiency and/or the immunostimulatoryproperties of the at least one RNA.

In a preferred embodiment, the at least one RNA of the RNA vaccineencoding at least one antigen in the inventive vaccine/inhibitorcombination may be formulated together with a cationic or polycationiccompound and/or with a polymeric carrier as defined herein. Accordingly,in a specific embodiment of the invention it is preferred that the atleast one RNA of the RNA vaccine encoding at least one antigen in theinventive vaccine/inhibitor combination is associated with or complexedwith a cationic or polycationic compound or a polymeric carrier asdefined herein, optionally in a weight ratio selected from a range ofabout 6:1 (w/w) to about 0.25:1 (w/w), more preferably from about 5:1(w/w) to about 0.5:1 (w/w), even more preferably of about 4:1 (w/w) toabout 1:1 (w/w) or of about 3:1 (w/w) to about 1:1 (w/w), and mostpreferably a ratio of about 3:1 (w/w) to about 2:1 (w/w) of RNA tocationic or polycationic compound and/or with a polymeric carrier; oroptionally in an N/P-ratio of at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.75, 1,1.5 or 2. Preferably, the N/P-ratio lies within a range of about 0.1,0.3, 0.4, 0.5, 0.75, 1.0, 1.5 or 2 to 20, preferably in a range of about0.2 (0.5 or 0.75 or 1.0) to 12, more preferably in an N/P-ratio of about0.4 (0.75 or 1.0) to 10, and even more preferably in an N/P ratio ofabout 0.4 (0.75 or 1.0) to 5. Most preferably the N/P ratio lies in aratio between 0.1 and 0.9.

In this context it is preferable that the cationic or polycationiccompound or the polymeric carrier used as carrier or complexation agentand the at least one RNA of the RNA vaccine encoding at least oneantigen in the inventive vaccine/inhibitor combination as defined hereinare provided in an N/P-ratio of at least about 1 or, preferably, of arange of about 1 to 20 for in vitro applications (e.g. in the case cellsextracted from the patient would be treated in vitro with the inventivepharmaceutical composition and subsequently administered to thepatient).

For in vivo applications, an N/P ratio of at least 0.1 (0.2, 0.3, 0.4,0.5, 0.6), preferably of a range of about 0.1 (0.2, 0.3, 0.4., 0.5, or0.6) to 1.5 is preferred. Even more preferred is an N/P ratio range of0.1 or 0.2 to 0.9 or an N/P ratio range of 0.5 to 0.9.

The N/P ratio significantly influences the surface charge of theresulting complex consisting of cationic or polycationic compounds or ofa polymeric carrier and a nucleic acid cargo e.g. the at least one RNAcoding for at least one antigen comprised in the RNA vaccine or of anadjuvant nucleic acid. Thus, it is preferable that the resultingpolymeric carrier cargo complex is positively charged for in vitroapplications and negatively or neutrally charged for in vivoapplications. The surface charge of the resulting polymeric carriercargo complex can be indicated as Zetapotential which may be measured byDoppler electrophoresis method using a Zetasizer Nano (MalvernInstruments, Malvern, UK).

The at least one RNA of the RNA vaccine encoding at least one antigen inthe inventive vaccine/inhibitor combination may also be associated witha vehicle, transfection or complexation agent for increasing thetransfection efficiency and/or the immunostimulatory properties of theat least one RNA.

In this context, it is particularly preferred that the at least one RNAof the RNA vaccine encoding at least one antigen in the inventivevaccine/inhibitor combination is complexed at least partially with acationic or polycationic compound and/or a polymeric carrier, preferablycationic proteins or peptides. Partially means that only a part of theat least one RNA is complexed with a cationic compound and that the restof the at least one RNA of the RNA vaccine is comprised in the inventivevaccine/inhibitor combination in uncomplexed form (“free” or “naked”).Preferably, the ratio of complexed RNA to: uncomplexed RNA in the RNAvaccine of the inventive vaccine/inhibitor combination is selected froma range of about 5:1 (w/w) to about 1:10 (w/w), more preferably from arange of about 4:1 (w/w) to about 1:8 (w/w), even more preferably from arange of about 3:1 (w/w) to about 1:5 (w/w) or 1:3 (w/w), and mostpreferably the ratio of complexed RNA to free RNA in the RNA vaccine ofthe inventive vaccine/inhibitor combination is selected from a ratio ofabout 1:1 (w/w).

The at least one complexed RNA in the RNA vaccine of the inventivevaccine/inhibitor combination, is preferably prepared according to afirst step by complexing the at least one RNA with a cationic orpolycationic compound and/or with a polymeric carrier, preferably asdefined herein, in a specific ratio to form a stable complex. In thiscontext, it is highly preferable, that no free cationic or polycationiccompound or polymeric carrier or only a negligibly small amount thereofremains in the component of the complexed RNA after complexing the RNA.Accordingly, the ratio of the RNA and the cationic or polycationiccompound and/or the polymeric carrier in the component of the complexedRNA is typically selected in a range that the RNA is entirely complexedand no free cationic or polycationic compound or polymeric carrier oronly a negligibly small amount thereof remains in the composition.

Preferably, the ratio of the at least one RNA (e.g. mRNA) comprising atleast one open reading frame coding for at least one antigen to thecationic or polycationic compound and/or the polymeric carrier,preferably as defined herein, is selected from a range of about 6:1(w/w) to about 0.25:1 (w/w), more preferably from about 5:1 (w/w) toabout 0.5:1 (w/w), even more preferably of about 4:1 (w/w) to about 1:1(w/w) or of about 3:1 (w/w) to about 1:1 (w/w), and most preferably aratio of about 3:1 (w/w) to about 2:1 (w/w). Alternatively, the ratio ofthe RNA to the cationic or polycationic compound and/or the polymericcarrier, preferably as defined herein, in the component of the complexedRNA, may also be calculated on the basis of the nitrogen/phosphate ratio(N/P-ratio) of the entire complex. In the context of the presentinvention, an N/P-ratio is preferably in the range of about 0.1-10,preferably in a range of about 0.3-4 and most preferably in a range ofabout 0.5-2 or 0.7-2 regarding the ratio of cationic or polycationiccompound and/or polymeric carrier: RNA, preferably as defined herein, inthe complex, and most preferably in a range of about 0.7-1.5, 0.5-1 or0.7-1, and even most preferably in a range of about 0.3-0.9 or 0.5-0.9preferably provided that the cationic or polycationic compound in thecomplex is a cationic or polycationic cationic or polycationic proteinor peptide and/or the polymeric carrier as defined above. In thisspecific embodiment, the complexed RNA is also emcompassed in the term“adjuvant component”.

In another embodiment, the at least one antigen-providing RNA in the RNAvaccine of the inventive vaccine/inhibitor combination as defined abovemay be formulated together with an adjuvant. Such an adjuvant may bepreferably a further nucleic acid that is not encoding a further antigenbut is able to stimulate an unspecific immune response, i.e. innateimmune response, by interacting with any part of the innate immunesystem. Such a nucleic acid stimulating an unspecific immune response istermed herein as “adjuvant nucleic acid”.

In this context, an adjuvant nucleic acid preferably comprises orconsists of an oligo- or a polynucleotide; more preferably an adjuvantnucleic acid comprising or consisting of an RNA or a DNA; even morepreferably such an adjuvant nucleic acid comprising or consisting of anRNA or a DNA being complexed with a cationic or polycationic compoundand/or with a polymeric carrier as defined herein; optionally in aweight ratio selected from a range of about 6:1 (w/w) to about 0.25:1(w/w), more preferably from about 5:1 (w/w) to about 0.5:1 (w/w), evenmore preferably of about 4:1 (w/w) to about 1:1 (w:w) or of about 3:1(w/w) to about 1:1 (w/w), and most preferably a ratio of about 3:1 (w/w)to about 2:1 (w/w) of adjuvant nucleic acid to cationic or polycationiccompound and/or with a polymeric carrier; or optionally in anitrogen/phosphate ratio of the cationic or polycationic compound and/orpolymeric carrier to adjuvant nucleic acid in the range of about 0.1-10,preferably in a range of about 0.3-4, most preferably in a range ofabout 0.7-1 or 0.5-1, and even most preferably in a range of about0.3-0.9 or 0.5-0.9. Such a complexed adjuvant nucleic acid is alsoencompassed in the term “adjuvant component”:

In the specific case that the induction of IFN-α is intended, an N/Pratio of at least 0.1 (0.2, 0.3, 0.4, 0.5, or 0.6) or an N/P ratio rangeof 0.1 to 1 is preferred or more preferred is an N/P ratio range of 0.1or 0.2 to 0.9 or an N/P ratio range of 0.5 to 0.9. Otherwise, if theinduction of TNFα would be intended, an N/P ratio of 1 to 20 isparticularly preferred.

In other words, the RNA vaccine of the vaccine/inhibitor combinationaccording to the invention may comprise the at least one RNA encoding atleast one antigen, and a further nucleic acid that is acting as anadjuvant which is called the adjuvant nucleic acid. Of course the RNAvaccine of the inventive vaccine/inhibitor combination is not limited tocomprise only one adjuvant nucleic acid but may comprise severaldifferent such nucleic acids. Both kinds of nucleic acid, theantigen-encoding RNA and the adjuvant nucleic acid, may be,independently from each other, complexed with a carrier as definedherein. Therefore, a cationic or polycationic compound and/or apolymeric carrier used to complex the at least one RNA of the RNAvaccine encoding at least one antigen or the adjuvant nucleic acid, maybe selected from any cationic or polycationic compound and/or polymericcarrier as defined herein.

In case the RNA vaccine of the inventive vaccine/inhibitor combination(or the inventive vaccine/inhibitor combination) comprises anantigen-providing RNA and additionally an adjuvant nucleic acid, theimmune response that is evoked by administration of such a vaccinecomprises activation of both parts of the immune system, the adaptiveimmune system as well as the innate immune system.

A substantial factor for a suitable adaptive immune response is thestimulation of different T cell sub-populations. T-lymphocytes aretypically divided into two sub-populations, the T-helper 1 cells, in thefollowing Th1-cells, and the T-helper 2 cells, in the followingTh2-cells, with which the immune system is capable of destroyingintracellular and extracellular pathogens (e.g. antigens). TherebyTh1-cells are responsible for intracellular pathogen destruction byassisting the cellular immune response by activation of macrophages andcytotoxic T cells. Th2-cells, on the other hand, are mainly forextracellular pathogen-elimination and promote the humoral immuneresponse by stimulation of B-cells for conversion into plasma cells andby formation of antibodies (e.g. against antigens). The two T-helpercell populations differ in the pattern of the effector proteins(cytokines) produced by them.

The Th1-cell/Th2-cell ratio is of great importance in the induction andmaintenance of an adaptive immune response. In connection with thepresent invention, the Th1-cell/Th2-cell ratio of the (adaptive) immuneresponse is preferably shifted in the direction towards the cellularresponse (Th1 response) and a cellular immune response is therebyinduced. Stimulation of this response of the adaptive immune system ismainly provoked by the translation of the antigen-providing RNA and theresulting presence of the peptide or protein antigens within theorganism.

The innate immune system which may support such an adaptive immuneresponse and which may induce or support a shift towards a Th1 responsemay be activated by ligands of Toll-like receptors (TLRs). TLRs are afamily of highly conserved pattern recognition receptor (PRR)polypeptides that recognize pathogen-associated molecular patterns(PAMPs) and play a critical role in innate immunity in mammals.Currently at least thirteen family members, designated TLR1-TLR13(Toll-like receptors: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8,TLR9, TLR10, TLR11, TLR12 or TLR13), have been identified. Furthermore,a number of specific TLR ligands have been identified. It was e.g. foundthat unmethylated bacterial DNA and synthetic analogs thereof (CpG DNA)are ligands for TLR9 (Hemmi H et al. (2000) Nature 408:740-5; Bauer S etal. (2001) Proc Natl. Acad. Sci. USA 98, 9237-42). Furthermore, it hasbeen reported that ligands for certain TLRs include certain nucleic acidmolecules and that certain types of RNA are immunostimulatory in asequence-independent or sequence-dependent manner, wherein these variousimmunostimulatory RNAs may e.g. stimulate TLR3, TLR7, or TLR8, orintracellular receptors such as RIG-I, MDA-5, etc.

In the context of the invention, the activation of the innate immunesystem can be provided by an adjuvant nucleic acid, preferably animmunostimulatory RNA (isRNA) as defined herein, comprised in theinventive vaccine/inhibitor combination, preferably comprised in the RNAvaccine.

According to the above, in a further preferred embodiment of theinvention, the RNA vaccine of the inventive vaccine/inhibitorcombination is formulated to comprise

-   -   a) said at least one RNA comprising at least one open reading        frame coding for at least one antigen; preferably in form of a        mono-, bi- or multicistronic RNA, optionally being stabilized,        optionally being optimized for translation and/or optionally        being complexed with a cationic or polycationic compound or a        polymeric carrier;    -   b) optionally an adjuvant component, comprising or consisting of        said at least one RNA comprising at least one open reading frame        coding for at least one antigen and/or at least one adjuvant        nucleic acid, complexed with a cationic or polycationic compound        and/or with a polymeric carrier, and    -   c) optionally a pharmaceutically acceptable carrier as defined        herein.

In this context, it is particularly preferred that the optionallycomprised adjuvant component comprises the same RNA as comprised in theRNA vaccine of the inventive vaccine/inhibitor combination asantigen-providing RNA e.g. mRNA coding for at least one antigen.

Furthermore, the RNA vaccine of the inventive vaccine/inhibitorcombination may comprise further components for facilitatingadministration and uptake of the components of the RNA vaccine. Suchfurther components may be an appropriate carrier or vehicle, or e.g.additional adjuvants for supporting any immune response as definedherein.

According to one further embodiment, the components of the RNA vaccinee.g. the at least one RNA coding for at least one antigen and anadjuvant component, of the inventive vaccine/inhibitor combination, maybe formulated together or separately in the same or differentcompositions.

As a second component, the inventive vaccine/inhibitor combinationincludes as inhibitor a composition comprising a PD-1 pathway inhibitortargeting any member of the PD-1 signaling pathway, preferably targetingPD-1, PD-L1 or PD-L2.

Programmed Death-1 (PD-1, PDCD1) is a type I transmembrane proteinbelonging to the extended CD28 family of T cell regulators. PD-1 lacksthe membrane-proximal cysteine residue required for homodimerization ofother members of the CD28 family. Structural and biochemical analysesshowed that PD-1 is monomeric in solution as well as on the cell surface(Okazaki and Honjo, 2007. Int Immunol. 19(7):813-24). PD-1 is expressedon activated T cells, B cells and monocytes. The broader expression ofPD-1 contrasts with restricted expression of other CD28 family membersto T cells, suggesting that PD-1 regulates a wider spectrum of immuneresponses compared with other CD28 family members.

PD-1 negatively regulates antigen receptor signaling by recruiting theprotein tyrosine phosphatase SHP-2 upon interacting with either of twoligands, PD-L1 or PD-L2.

PD-L1 (B7-H1, CD274) and PD-L2 (B7-DC, CD273) are type I transmembraneglycoproteins composed of IgC-and IgV-type extracellular domains. PD-L1and PD-L2 share 40% amino acid identity while human and mouse orthologsof PD-L1 and PD-L2 share 70% amino acid identity. Both PD-L1 and PD-L2have short cytoplasmic tails with no known motif for signaltransduction, suggesting that these ligands do not transduce any signalupon interaction with PD-1.

The interaction of PD-L1 and PD-1 provides a crucial negativeco-stimulatory signal to T cells and functions as cell death inducer.Interaction between low concentration of PD-1 and PD-L1 leads to thetransmission of an inhibitory signal that inhibits the proliferation ofantigen-specific CD8+ cells. At higher concentrations this interactiondoes not inhibit T cell proliferation but reduces the production ofmultiple cytokines. Thus, binding to PD-L1 can antagonize the B7-CD28signal when antigenic stimulation is weak and plays a key role indownregulating T cell responses.

The role of PD-1 and PD-1 ligands in inhibiting T cell activation andproliferation suggested that these proteins may serve as therapeutictargets for treatments of inflammation, cancer or infectious diseases.Depending on the desired therapeutic outcome, an up- or down-modulationof the PD-1 pathway is required. Up-modulation of the immune system isparticularly required in the treatment of cancers and chronicinfections. This can be achieved for example by PD-1 blockade orinhibiting the PD-1 pathway. Inhibition of the PD-1 pathway can beachieved, for example, by an antibody directed at PD-1 or a PD-1 ligand.In this context the PD-1 pathway inhibitor may remove T cell dysfunctionresulting from PD-1 signaling and thereby restore or enhance T cellfunction (e.g. proliferation, cytokine production, target cell killing).In addition, anergic T cells which are unresponsive to antigenstimulation may be reactivated.

In the context of the present invention, the PD-1 pathway inhibitor maybe in specific embodiments an antibody, particularly an antagonisticantibody or a nucleic acid-encoded antibody (intrabody), an siRNA, anantisense RNA, a protein comprising (or a nucleic acid coding for) anamino acid sequence capable of binding to PD-1 but preventing PD-1signaling (e.g. a fusion protein of a fragment of PD-L1 or PD-L2 and theFc part of an immunoglobulin), a soluble protein (or a nucleic acidcoding for a soluble protein) competing with membrane-bound PD-1 forbinding of its ligands PD-L1 and PD-L2; or a small molecule inhibitorcapable of inhibiting PD-1 pathway signaling.

Therefore, in a preferred embodiment of the present invention, the PD-1pathway inhibitor is an antibody (or a nucleic acid coding for anantibody) directed against PD-1, preferably an antibody specificallybinding to the extracellular domain of PD-1 and thereby inhibiting PD-1signaling. Preferably, such an antagonistic antibody binds close to thePD-L1 binding site on PD-1, thus inhibiting the binding of PD-L1 toPD-1.

Particularly preferred are the anti-PD1 antibodies Nivolumab(MDX-1106/BMS-936558/ONO-4538), (Brahmer et al., 2010. J Clin Oncol.28(19):3167-75; PMID: 20516446); Pidilizumab (CT-011), (Berger et al.,2008. Clin Cancer Res. 14(10):3044-51; PMID:

18483370); and MK-3475 (SCH 900475).

In a further preferred embodiment, the PD-1 pathway inhibitor is anantibody (or a nucleic acid coding for an antibody) directed against aPD-1 ligand, preferably an antibody specifically binding to theextracellular domain of the PD-1 or PD-2 ligand. Preferably, suchantibody binds proximal to and disruptive of the PD-1 or PD-2 bindingsite on the ligand.

Particularly preferred are the anti-PD-L1 antibodies MDX-1105/BMS-936559(Brahmer et al. 2012. N Engl J Med. 366(26):2455-65; PMID: 22658128);MPDL3280A/RG7446, or MEDI4736.

In a further preferred embodiment, the PD-1 pathway inhibitor is aprotein comprising (or a nucleic acid coding for) an amino acid sequencecapable of binding to PD-1 but preventing PD-1 pathway signaling,particularly preferred in this context is a fusion protein of a fragmentof PD-L1 or PD-L2 ligand.

In this context, a particularly preferred embodiment is a fusion proteincomprising the extracellular domain of PD-L1 or PD-L2 or a fragmentthereof capable of binding to PD-1 and an Fc portion of animmunoglobulin. An example of such a fusion protein is represented byAMP-224 (extracellular domain of murine PD-L2/B7-DC fused to theunmodified Fc portion of murine IgG2a protein; Mkrtichyan et al., 2012.J Immunol. 189(5):2338-47; PMID: 22837483).

In the context of the present invention, the administration of thevaccine and the inhibitor may occur either simultaneously or timelystaggered, either at the same site of administration or at differentsites of administration, as further outlined below.

To ensure that the separate mechanisms elicited by the RNA vaccine andthe PD-1 pathway inhibitor are not negatively influenced by each other,the PD-1 pathway inhibitor and the RNA vaccine are preferablyadministered separated in time (in a time-staggered manner), i.e.sequentially, and/or are administered at different administration sites.This means that the RNA vaccine may be administrated e.g. prior,concurrent or subsequent to the PD-1 pathway inhibitor, or vice versa.Alternatively or additionally, the RNA vaccine and the PD-1 pathwayinhibitor may be administered at different administration sites, or atthe same administration site, preferably, when administered in a timestaggered manner. According to a particularly preferred embodiment, theRNA vaccine is to be administered first and the PD-1 pathway inhibitoris to be administered subsequent to the RNA vaccine. This procedureensures that the immune cells such as antigen-presenting cells and Tcells have already encountered the antigen before the immune system isstimulated by inhibition of the PD-1 pathway, even though a concurrentadministration or an administration, wherein the PD-1 pathway inhibitoris to be administered prior to the RNA vaccine, may lead to the same orat least comparable results.

Accordingly, in a further embodiment, the inventive vaccine/inhibitorcombination furthermore comprises a pharmaceutically acceptable carrierand/or vehicle.

Such a pharmaceutically acceptable carrier typically includes the liquidor non-liquid basis of a composition comprising the components of theinventive vaccine/inhibitor combination. If the composition is providedin liquid form, the carrier will preferably be pyrogen-free water;isotonic saline or buffered (aqueous) solutions, e.g. phosphate, citrateetc. buffered solutions. The injection buffer may be hypertonic,isotonic or hypotonic with reference to the specific reference medium,i.e. the buffer may have a higher, identical or lower salt content withreference to the specific reference medium, wherein preferably suchconcentrations of the afore mentioned salts may be used, which do notlead to damage of cells due to osmosis or other concentration effects.Reference media are e.g. liquids occurring in “in vivo” methods, such asblood, lymph, cytosolic liquids, or other body liquids, or e.g. liquids,which may be used as reference media in “in vitro” methods, such ascommon buffers or liquids. Such common buffers or liquids are known to askilled person. Ringer-Lactate solution is particularly preferred as aliquid basis.

However, one or more compatible solid or liquid fillers or diluents orencapsulating compounds, which are suitable for administration to apatient to be treated, may be used as well for the vaccine/inhibitorcombination according to the invention. The term “compatible” as usedhere means that these constituents of the vaccine/inhibitor combinationare capable of being mixed with the RNA vaccine and/or the PD-1 pathwayinhibitor in such a manner that no interaction occurs which wouldsubstantially reduce the pharmaceutical effectiveness of thevaccine/inhibitor combination under typical use conditions.

Furthermore, the inventive vaccine/inhibitor combination may compriseone or more additional adjuvants which are suitable to initiate orincrease an immune response of the innate immune system, i.e. anon-specific immune response, particularly by binding topathogen-associated molecular patterns (PAMPs). With other words, whenadministered, the RNA vaccine preferably elicits an innate immuneresponse due to the adjuvant, optionally contained therein.Nevertheless, the adjuvant may also be part of another component of theinventive vaccine/inhibitor combination than the RNA vaccine.Preferably, such an adjuvant may be selected from an adjuvant known to askilled person and suitable for the present case, i.e. supporting theinduction of an innate immune response in a mammal, e.g. an adjuvantnucleic acid or an adjuvant component as defined above or an adjuvant asdefined in the following.

Therefore, such an adjuvant may also be selected from any adjuvant knownto a skilled person and suitable for the present case, i.e. supportingthe induction of an innate immune response in a mammal and/or suitablefor depot and delivery of the components of the inventivevaccine/inhibitor combination. Preferred as adjuvants suitable for depotand delivery are cationic or polycationic compounds as defined above.Likewise, the adjuvant may be selected from the group consisting of,e.g., cationic or polycationic compounds as defined above, fromchitosan, TDM, MDP, muramyl dipeptide, pluronics, alum solution,aluminium hydroxide, ADJUMER™ (polyphosphazene); aluminium phosphategel; glucans from algae; algammulin; aluminium hydroxide gel (alum);highly protein-adsorbing aluminium hydroxide gel; low viscosityaluminium hydroxide gel; AF or SPT (emulsion of squalane (5%), Tween 80(0.2%), Pluronic L121 (1.25%), phosphate-buffered saline, pH 7.4);AVRIDINE™ (propanediamine); BAY R1005™((N-(2-deoxy-2-L-leucylaminob-D-glucopyranosyl)-N-octadecyl-dodecanoyl-amidehydroacetate); CALCITRIOL™ (1-alpha,25-dihydroxy-vitamin D3); calciumphosphate gel; CAP™ (calcium phosphate nanoparticles); choleraholotoxin, cholera-toxin-Al-protein-A-D-fragment fusion protein,sub-unit B of the cholera toxin; CRL 1005 (block copolymer P1205);cytokine-containing liposomes; DDA (dimethyldioctadecylammoniumbromide); DHEA (dehydroepiandrosterone); DMPC(dimyristoylphosphatidylcholine); DMPG(dimyristoylphosphatidylglycerol); DOC/alum complex (deoxycholic acidsodium salt); Freund's complete adjuvant; Freund's incomplete adjuvant;gamma inulin; Gerbu adjuvant (mixture of: i)N-acetylglucosaminyl-(P1-4)-N-acetylmuramyl-L-alanyl-D35 glutamine(GMDP), ii) dimethyldioctadecylammonium chloride (DDA), iii)zinc-L-proline salt complex (ZnPro-8); GM-CSF); GMDP(N-acetylglucosaminyl-(b1-4)-N-acetylmuramyl-L47 alanyl-D-isoglutamine);imiquimod (1-(2-methypropyl)-1H-imidazo[4,5-c]quinoline-4-amine);ImmTher™(N-acetylglucosaminyl-N-acetylmuramyl-L-Ala-D-isoGlu-L-Ala-glyceroldipalmitate); DRVs (immunoliposomes prepared fromdehydration-rehydration vesicles); interferongamma; interleukin-1beta;interleukin-2; interleukin-7; interleukin-12; ISCOMS™; ISCOPREP 7.0.3.™;liposomes; LOXORIBINE™ (7-allyl-8-oxoguanosine); LT 5 oral adjuvant (E.coli labile enterotoxin-protoxin); microspheres and microparticles ofany composition; MF59™; (squalenewater emulsion); MONTANIDE ISA 51™(purified incomplete Freund's adjuvant); MONTANIDE ISA 720™(metabolisable oil adjuvant); MPL™ (3-Q-desacyl-4′-monophosphoryl lipidA); MTP-PE and MTP-PE liposomes((N-acetyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1,2-dipalmitoyl-sn-glycero-3-(hydroxyphosphoryloxy))-ethylamide,monosodium salt); MURAMETIDE™ (Nac-Mur-L-Ala-D-Gln-OCH3); MURAPALMITINE™and DMURAPALMITINE™ (Nac-Mur-L-Thr-D-isoGln-sn-glyceroldipalmitoyl);NAGO (neuraminidase-galactose oxidase); nanospheres or nanoparticles ofany composition; NISVs (non-ionic surfactant vesicles); PLEURAN™(□-glucan); PLGA, PGA and PLA (homo- and co-polymers of lactic acid andglycolic acid; microspheres/nanospheres); PLURONIC L121™; PMMA(polymethylmethacrylate); PODDS™ (proteinoid microspheres); polyethylenecarbamate derivatives; poly-rA: poly-rU (polyadenylic acid-polyuridylicacid complex); polysorbate 80 (Tween 80); protein cochleates (AvantiPolar Lipids, Inc., Alabaster, AL); STIMULON™ (QS-21); Quil-A (Quil-Asaponin); S-28463(4-amino-otec-dimethyl-2-ethoxymethyl-1H-imidazo[4,5-c]quinoline-1-ethanol);SAF-1™ (“Syntex adjuvant formulation”); Sendai proteoliposomes andSendai containing lipid matrices; Span-85 (sorbitan trioleate); Specol(emulsion of Marcol 52, Span 85 and Tween 85); squalene or Robane(2,6,10,15,19,23-hexamethyltetracosan and 2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexane); stearyltyrosine(octadecyltyrosine hydrochloride); Theramid®(N-acetylglucosaminyl-N-acetylmuramyl-L-Ala-D-isoGlu-L-Aladipalmitoxypropylamide);Theronyl-MDP (Termurtide™ or [thr 1]-MDP;N-acetylmuramyl-Lthreonyl-D-isoglutamine); Ty particles (Ty-VLPs orvirus-like particles); Walter-Reed liposomes (liposomes containing lipidA adsorbed on aluminium hydroxide), and lipopeptides, including Pam3Cys,in particular aluminium salts, such as Adju-phos, Alhydrogel,Rehydragel; emulsions, including CFA, SAF, IFA, MF59, Provax, TiterMax,Montanide, Vaxfectin; copolymers, including Optivax (CRL1005), L121,Poloaxmer4010), etc.; liposomes, including Stealth, cochleates,including BIORAL; plant derived adjuvants, including QS21, Quil A,Iscomatrix, ISCOM; adjuvants suitable for costimulation includingTomatine, biopolymers, including PLG, PMM, Inulin, microbe derivedadjuvants, including Romurtide, DETOX, MPL, CWS, Mannose, CpG nucleicacid sequences, CpG7909, ligands of human TLR 1-10, ligands of murineTLR 1-13, ISS-1018, 35 IC31, Imidazoquinolines, Ampligen, Ribi529,IMOxine, IRIVs, VLPs, cholera toxin, heat-labile toxin, Pam3Cys,Flagellin, GPI anchor, LNFPIII/Lewis X, antimicrobial peptides,UC-1V150, RSV fusion protein, cdiGMP; and adjuvants suitable asantagonists including CGRP neuropeptide.

An adjuvant is preferably selected from adjuvants, which supportinduction of a Th1-immune response or maturation of naïve T-cells, suchas GM-CSF, IL-12, IFNg, any adjuvant nucleic acid as defined above,preferably an immunostimulatory RNA, CpG DNA, etc.

In a further preferred embodiment, it is also possible that theinventive vaccine/inhibitor combination contains besides theantigen-providing RNA and the PD-1 pathway inhibitor further componentswhich are selected from the group comprising: further antigens orfurther antigen-providing nucleic acids; a further immunotherapeuticagent; one or more auxiliary substances; or any further compound, whichis known to be immunostimulating due to its binding affinity (asligands) to human Toll-like receptors; and/or an adjuvant nucleic acid,preferably an immunostimulatory RNA (isRNA).

Accordingly, in another preferred embodiment, the inventivevaccine/inhibitor combination furthermore comprises at least oneadjuvant, an auxiliary substance selected from lipopolysaccharides,TNF-alpha, CD40 ligand, or cytokines, monokines, lymphokines,interleukins or chemokines, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,IL-8, IL-9, IL-10, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18,IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28,IL-29, IL-30, IL-31, IL-32, IL-33, IFN-alpha, IFN-beta, IFN-gamma,GM-CSF, G-CSF, M-CSF, LT-beta, TNF-alpha, growth factors, and hGH, aligand of human Toll-like receptor TLR1, TLR2, TLR3, TLR4, TLR5, TLR6,TLR7, TLR8, TLR9, TLR10, a ligand of murine Toll-like receptor TLR1,TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12 orTLR13, a ligand of a NOD-like receptor, a ligand of a RIG-I likereceptor, an adjuvant nucleic acid, an immunostimulatory RNA (isRNA), aCpG-DNA, an antibacterial agent, or an anti-viral agent.

The vaccine/inhibitor combination as defined according to the presentinvention may furthermore comprise further additives or additionalcompounds. Further additives which may be included in the inventivevaccine/inhibitor combination, such as in the RNA vaccine and/or thecomposition comprising a PD-1 pathway inhibitor, are emulsifiers, suchas, for example, Tween®; wetting agents, such as, for example, sodiumlauryl sulfate; colouring agents; taste-imparting agents, pharmaceuticalcarriers; tablet-forming agents; stabilizers; antioxidants;preservatives, RNase inhibitors and/or an anti-bacterial agent or ananti-viral agent.

The inventive vaccine/inhibitor combination typically comprises a “safeand effective amount” of the components of the inventivevaccine/inhibitor combination as defined herein. As used herein, a “safeand effective amount” preferably means an amount of the components,preferably of the at least one RNA encoding at least one antigen and thePD-1 pathway inhibitor, that is sufficient to significantly induce apositive modification or prevention of a disease or disorder as definedherein. At the same time, however, a “safe and effective amount” issmall enough to avoid serious side-effects and to permit a sensiblerelationship between advantage and risk. The determination of theselimits typically lies within the scope of sensible medical judgment.

The inventive vaccine/inhibitor combination may be administered orally,parenterally, by inhalation spray, topically, rectally, nasally,buccally, vaginally or via an implanted reservoir.

The term parenteral as used herein includes subcutaneous, intravenous,intramuscular, intra-articular, intra-nodal, intra-synovial,intrasternal, intrathecal, intrahepatic, intralesional, intracranial,transdermal, intradermal, intrapulmonal, intraperitoneal, intracardial,intraarterial, and sublingual injection or infusion techniques.Preferably the RNA vaccine is administered by intradermal orintramuscular application and the PD-1 pathway inhibitor is preferablyadministered by intramuscular or intraperitoneal injection, morepreferably by intravenous infusion, in case it is in form of anantibody.

According to a further aspect, the object underlying the presentinvention is solved by a pharmaceutical composition comprising a vaccineand an inhibitor, in particular as vaccine an RNA vaccine comprising atleast one RNA comprising at least one open reading frame coding for atleast one antigen and as an inhibitor a composition comprising a PD-1pathway inhibitor, both preferably as defined above. Likewise, thepharmaceutical composition is preferably formulated and administered asdefined above for the components of the inventive vaccine/inhibitorcombination. Such a pharmaceutical composition may further comprise anyingredient as defined above for the inventive vaccine/inhibitorcombination.

Accordingly, the combination of the RNA vaccine and the PD-1 pathwayinhibitor as defined according to the present invention may occur eitheras one composition, e.g. the pharmaceutical composition according to thepresent invention, or may occur in more than one compositions, e.g. as akit of parts, wherein the different components form different parts ofsuch kit of parts. These different components, such as the vaccine andthe inhibitor, may be formulated each as a pharmaceutical composition oras a composition as defined above. Preferably, each of the differentparts of the kit comprises a different component, e.g. one partcomprises the RNA vaccine as defined herein, one further part comprisesthe PD-1 pathway inhibitor as defined herein, etc.

Therefore, according to a further aspect, the present invention alsoprovides kits, particularly kits of parts. Such kits, particularly kitsof parts, typically comprise as components alone or in combination withfurther components as defined herein an RNA vaccine comprising at leastone RNA comprising at least one open reading frame coding for at leastone antigen and, preferably in a different part of the kit, a PD-1pathway inhibitor as defined herein. The inventive vaccine/inhibitorcombination as defined herein, optionally in combination with furthercomponents as defined herein, such as an additional adjuvant, may occurin one or different parts of the kit. As an example, e.g. at least onepart of the kit may comprise the RNA vaccine comprising at least one RNAencoding at least one antigen as defined herein, at least one furtherpart of the kit may comprise the PD-1 pathway inhibitor as definedherein, and optionally at least one further part of the kit may comprisean additional adjuvant as described herein. The kit or kit of parts mayfurthermore contain technical instructions with information on theadministration and dosage of the inventive vaccine/inhibitorcombination, the inventive pharmaceutical composition or of any of itscomponents or parts.

The inventive vaccine/inhibitor combination, the inventivepharmaceutical composition or the inventive kit of parts comprising anRNA vaccine and a PD-1 pathway inhibitor may be used for human and alsofor veterinary medical purposes, preferably for human medical purposes.

Therefore, according to a further aspect, the present invention isdirected to the first medical use of the inventive vaccine/inhibitorcombination, the inventive pharmaceutical composition and the inventivekit of parts comprising an RNA vaccine and a PD-1 pathway inhibitor asdefined herein. Accordingly, the inventive vaccine/inhibitorcombination, the inventive pharmaceutical composition and the inventivekit of parts comprising an RNA vaccine and a PD-1 pathway inhibitor asdefined herein may be used as a medicament.

According to another aspect, the present invention is directed to thesecond medical use of the inventive vaccine/inhibitor combination, theinventive pharmaceutical composition and the inventive kit of partscomprising an RNA vaccine and a PD-1 pathway inhibitor as definedherein. Thus, the inventive vaccine/inhibitor combination, the inventivepharmaceutical composition and the inventive kit of parts comprising anRNA vaccine and a PD-1 pathway inhibitor as defined herein may be usedfor the treatment and/or amelioration of various diseases, particularlyof cancer and tumor diseases and infectious diseases as defined herein.

Cancer or tumour diseases in this context preferably include e.g.melanomas, malignant melanomas, colon carcinomas, lymphomas, sarcomas,blastomas, renal carcinomas, gastrointestinal tumors, gliomas, prostatetumors, bladder cancer, rectal tumors, stomach cancer, oesophagealcancer, pancreatic cancer, liver cancer, mammary carcinomas (=breastcancer), uterine cancer, cervical cancer, acute myeloid leukaemia (AML),acute lymphoid leukaemia (ALL), chronic myeloid leukaemia (CML), chroniclymphocytic leukaemia (CLL), hepatomas, various virus-induced tumorssuch as, for example, papilloma virus-induced carcinomas (e.g. cervicalcarcinoma=cervical cancer), adenocarcinomas, herpes virus-induced tumors(e.g. Burkitt's lymphoma, EBV-induced B-cell lymphoma), heptatitisB-induced tumors (hepatocell carcinomas), HTLV-1- and HTLV-2-inducedlymphomas, acoustic neuroma, lung carcinomas (=lung cancer=bronchialcarcinoma), small-cell lung carcinomas, pharyngeal cancer, analcarcinoma, glioblastoma, rectal carcinoma, astrocytoma, brain tumors,retinoblastoma, basalioma, brain metastases, medulloblastomas, vaginalcancer, pancreatic cancer, testicular cancer, Hodgkin's syndrome,meningiomas, Schneeberger disease, hypophysis tumor, Mycosis fungoides,carcinoids, neurinoma, spinalioma, Burkitt's lymphoma, laryngeal cancer,renal cancer, thymoma, corpus carcinoma, bone cancer, non-Hodgkin'slymphomas, urethral cancer, CUP syndrome, head/neck tumors,oligodendroglioma, vulval cancer, intestinal cancer, colon carcinoma,oesophageal carcinoma (=oesophageal cancer), wart involvement, tumors ofthe small intestine, craniopharyngeomas, ovarian carcinoma, genitaltumors, ovarian cancer (=ovarian carcinoma), pancreatic carcinoma(=pancreatic cancer), endometrial carcinoma, liver metastases, penilecancer, tongue cancer, gall bladder cancer, leukaemia, plasmocytoma, lidtumor, prostate cancer (=prostate tumors), etc.

In specific embodiments the treatment of lung cancer (e.g. non-smallcell lung cancer or small cell lung cancer) or prostate cancer isparticularly preferred.

Infectious diseases in this context, preferably includes viral,bacterial or protozoological infectious diseases. Such infectiousdiseases, preferably (viral, bacterial or protozoological) infectiousdiseases, are typically selected from the list consisting ofAcinetobacter infections, African sleeping sickness (Africantrypanosomiasis), AIDS (Acquired immunodeficiency syndrome), Amoebiasis,Anaplasmosis, Anthrax, Appendicitis, Arcanobacterium haemolyticuminfections, Argentine hemorrhagic fever, Ascariasis, Aspergillosis,Astrovirus infections, Athlete's foot, Babesiosis, Bacillus cereusinfections, Bacterial meningitis, Bacterial pneumonia, Bacterialvaginosis (BV), Bacteroides infections, Balantidiasis, Baylisascarisinfections, Bilharziosis, BK virus infections, Black piedra,Blastocystis hominis infections, Blastomycosis, Bolivian hemorrhagicfever, Borrelia infectionss (Borreliosis), Botulism (and Infantbotulism), Bovine tapeworm, Brazilian hemorrhagic fever, Brucellosis,Burkholderia infections, Buruli ulcer, Calicivirus infections (Norovirusand Sapovirus), Campylobacteriosis, Candidiasis (Candidosis), Caninetapeworm infections, Cat-scratch disease, Chagas Disease (Americantrypanosomiasis), Chancroid, Chickenpox, Chlamydia infections, Chlamydiatrachomatis infections, Chlamydophila pneumoniae infections, Cholera,Chromoblastomycosis, Climatic bubo, Clonorchiasis, Clostridium difficileinfections, Coccidioidomycosis, Cold, Colorado tick fever (CTF), Commoncold (Acute viral rhinopharyngitis; Acute coryza), Condyloma acuminata,Conjunctivitis, Creutzfeldt-Jakob disease (CJD), Crimean-Congohemorrhagic fever (CCHF), Cryptococcosis, Cryptosporidiosis, Cutaneouslarva migrans (CLM), Cutaneous Leishmaniosis, Cyclosporiasis,Cysticercosis, Cytomegalovirus infections, Dengue fever,Dermatophytosis, Dientamoebiasis, Diphtheria, Diphyllobothriasis,Donavanosis, Dracunculiasis, Early summer meningoencephalitis (FSME),Ebola hemorrhagic fever, Echinococcosis, Ehrlichiosis, Enterobiasis(Pinworm infections), Enterococcus infections, Enterovirus infections,Epidemic typhus, Epiglottitis, Epstein-Barr Virus InfectiousMononucleosis, Erythema infectiosum (Fifth disease), Exanthem subitum,Fasciolopsiasis, Fasciolosis, Fatal familial insomnia (FFI), Fifthdisease, Filariasis, Fish poisoning (Ciguatera), Fish tapeworm, Flu,Food poisoning by Clostridium perfringens, Fox tapeworm, Free-livingamebic infections, Fusobacterium infections, Gas gangrene, Geotrichosis,Gerstmann-Sträussler-Scheinker syndrome (GSS), Giardiasis, Glanders,Gnathostomiasis, Gonorrhea, Granuloma inguinale (Donovanosis), Group Astreptococcal infections, Group B streptococcal infections, Haemophilusinfluenzae infections, Hand foot and mouth disease (HFMD), HantavirusPulmonary Syndrome (HPS), Helicobacter pylori infections,Hemolytic-uremic syndrome (HUS), Hemorrhagic fever with renal syndrome(HFRS), Henipavirus infections, Hepatitis A, Hepatitis B, Hepatitis C,Hepatitis D, Hepatitis E, Herpes simplex, Herpes simplex type I, Herpessimplex type II, Herpes zoster, Histoplasmosis, Hollow warts, Hookworminfections, Human bocavirus infections, Human ewingii ehrlichiosis,Human granulocytic anaplasmosis (HGA), Human metapneumovirus infections,Human monocytic ehrlichiosis, Human papillomavirus (HPV) infections,Human parainfluenza virus infections, Hymenolepiasis, Influenza,Isosporiasis, Japanese encephalitis, Kawasaki disease, Keratitis,Kingella kingae infections, Kuru, Lambliasis (Giardiasis), Lassa fever,Legionellosis (Legionnaires' disease, Pontiac fever), Leishmaniasis,Leprosy, Leptospirosis, Lice, Listeriosis, Lyme borreliosis, Lymedisease, Lymphatic filariasis (Elephantiasis), Lymphocyticchoriomeningitis, Malaria, Marburg hemorrhagic fever (MHF), Marburgvirus, Measles, Melioidosis (Whitmore's disease), Meningitis,Meningococcal disease, Metagonimiasis, Microsporidiosis, Miniaturetapeworm, Miscarriage (prostate inflammation), Molluscum contagiosum(MC), Mononucleosis, Mumps, Murine typhus (Endemic typhus), Mycetoma,Mycoplasma hominis, Mycoplasma pneumonia, Myiasis, Nappy/diaperdermatitis, Neonatal conjunctivitis (Ophthalmia neonatorum), Neonatalsepsis (Chorioamnionitis), Nocardiosis, Noma, Norwalk virus infections,Onchocerciasis (River blindness), Osteomyelitis, Otitis media,Paracoccidioidomycosis (South American blastomycosis), Paragonimiasis,Paratyphus, Pasteurellosis, Pediculosis capitis (Head lice), Pediculosiscorporis (Body lice), Pediculosis pubis (Pubic lice, Crab lice), Pelvicinflammatory disease (PID), Pertussis (Whooping cough), Pfeiffer'sglandular fever, Plague, Pneumococcal infections, Pneumocystis pneumonia(PCP), Pneumonia, Polio (childhood lameness), Poliomyelitis, Porcinetapeworm, Prevotella infections, Primary amoebic meningoencephalitis(PAM), Progressive multifocal leukoencephalopathy, Pseudo-croup,Psittacosis, Q fever, Rabbit fever, Rabies, Rat-bite fever, Reiter'ssyndrome, Respiratory syncytial virus infections (RSV),Rhinosporidiosis, Rhinovirus infections, Rickettsial infections,Rickettsialpox, Rift Valley fever (RVF), Rocky mountain spotted fever(RMSF), Rotavirus infections, Rubella, Salmonella paratyphus, Salmonellatyphus, Salmonellosis, SARS (Severe Acute Respiratory Syndrome),Scabies, Scarlet fever, Schistosomiasis (Bilharziosis), Scrub typhus,Sepsis, Shigellosis (Bacillary dysentery), Shingles, Smallpox (Variola),Soft chancre, Sporotrichosis, Staphylococcal food poisoning,Staphylococcal infections, Strongyloidiasis, Syphilis, Taeniasis,Tetanus, Three-day fever, Tick-borne encephalitis, Tinea barbae(Barber's itch), Tinea capitis (Ringworm of the Scalp), Tinea corporis(Ringworm of the Body), Tinea cruris (Jock itch), Tinea manuum (Ringwormof the Hand), Tinea nigra, Tinea pedis (Athlete's foot), Tinea unguium(Onychomycosis), Tinea versicolor (Pityriasis versicolor), Toxocariasis(Ocular Larva Migrans (OLM) and Visceral Larva Migrans (VLM)),Toxoplasmosis, Trichinellosis, Trichomoniasis, Trichuriasis (Whipworminfections), Tripper, Trypanosomiasis (sleeping sickness), Tsutsugamushidisease, Tuberculosis, Tularemia, Typhus, Typhus fever, Ureaplasmaurealyticum infections, Vaginitis (Colpitis), Variant Creutzfeldt-Jakobdisease (vCJD, nvCJD), Venezuelan equine encephalitis, Venezuelanhemorrhagic fever, Viral pneumonia, Visceral Leishmaniosis, Warts, WestNile Fever, Western equine encephalitis, White piedra (Tinea blanca),Whooping cough, Yeast fungus spots, Yellow fever, Yersiniapseudotuberculosis infections, Yersiniosis, and Zygomycosis.

Particularly preferred are diseases, particularly cancer or tumordiseases, which are associated with an overexpression of at least onemember of the PD-1 pathway, preferably PD-1 receptor and/or its ligandsPD-L1 and PD-L2.

Particularly preferred in this context is the treatment of melanoma,glioblastoma, and carcinomas of the pancreas, lung, breast, colon,ovary, and renal cells, urothelial cancers, squamous cell carcinomas ofthe head and neck and hepatocellular carcinoma which are associated withan overexpression of PD-L1. Most particularly preferred is the treatmentof non-small cell lung cancer (NSCLC) or small cell lung cancerassociated with an overexpression of any member of the PD-1 pathway,particularly of PD-1 or PD-L1.

In another embodiment the treatment of cancer or tumor diseasesassociated with no or low expression of at least one member of the PD-1pathway, preferably PD-1 receptor and/or its ligands PD-L1 and PD-L2 isparticularly preferred. In this context the RNA vaccine of the inventivevaccine/inhibitor combination is able to induce the expression of atleast one member of the PD-1 pathway, e.g. PD-L1 in the patient to betreated and therefore enables the therapeutic activity of the PD-1inhibitor.

According to another aspect, the present invention provides a PD-1pathway inhibitor as defined above, for use in therapy in combinationwith an RNA vaccine comprising at least one RNA comprising at least oneopen reading frame coding for at least one antigen as defined above, forexample, for use in a method of treatment or prevention of tumor and/orcancer diseases or infectious diseases as defined herein.

According to yet another aspect, the present invention provides an RNAvaccine comprising at least one RNA comprising at least one open readingframe coding for at least one antigen as defined above, for use intherapy in combination with a PD-1 pathway inhibitor as defined above,for example, for use in a method of treatment or prevention of tumorand/or cancer diseases or infectious diseases as defined herein.

Furthermore, the present invention provides in a further aspect a methodfor transfecting and/or treating a cell, a tissue or an organism,thereby applying or administering the inventive vaccine/inhibitorcombination particularly for therapeutic purposes. In this context,typically after preparing the inventive vaccine/inhibitor combination,the inventive vaccine/inhibitor combination is preferably administeredto a cell, a tissue or an organism, preferably using any of theadministration modes as described herein. The method for transfectingand/or treating a cell may be carried out in vitro, in vivo or ex vivo.

Therefore, the invention also provides a method of treatment comprisingadministering to a subject in need thereof a therapeutically effectiveamount of an RNA vaccine comprising at least one RNA comprising at leastone open reading frame coding for at least one antigen as defined abovein combination with a composition comprising a PD-1 pathway inhibitor asdefined above.

In a preferred embodiment, the method comprises the in vitrotransfection of isolated cells. The cells used therefore are preferablyhuman or animal cells, particularly cells of a primary cell culture,which are then retransferred to a human or animal. Prior totransfection, these cells are typically isolated from the patient to betreated and cultivated.

In a further preferred embodiment, the method of treatment does notcomprise the in vitro transfection of isolated cells. In this case, theRNA vaccine as defined above administered to the subject in need thereofdoes not comprise isolated cells transfected with the at least one RNAcomprising at least one open reading frame coding for at least oneantigen comprised in the RNA vaccine.

In the present invention, if not otherwise indicated, different featuresof alternatives and embodiments may be combined with each other. In thecontext of the present invention, term “comprising” may be substitutedwith the term “consisting of”, where applicable.

BRIEF DESCRIPTION OF THE FIGURES

The figures shown in the following are merely illustrative and shalldescribe the present invention in a further way. These figures shall notbe construed to limit the present invention thereto.

FIG. 1: The RNA vaccine (OVA-RNActive R1710) acts synergistically withanti-PD-1 antibody. C57 BL/6 mice were challenged subcutaneously with3×10⁵ syngenic E.G.7-OVA tumor cells on day 0 and then treated witheither OVA RNActive vaccine (32 μg) alone or in combination withanti-PD-1 antibody or control IgG (100 μg i.p.) in accordance with theindicated schedule.

FIG. 2: Survival proportions of mice bearing E.G.7-OVA tumors treatedwith different therapies. According to Example 2, mice were treated witheither OVA-RNActive vaccine or anti-PD-1 antibody alone or incombination.

FIG. 3: G/C optimized mRNA sequence of R1710 coding for Gallus gallusovalbumin as comprised in the OVA-RNActive vaccine.

EXAMPLES

The examples shown in the following are merely illustrative and shalldescribe the present invention in a further way. These examples shallnot be construed to limit the present invention thereto.

Example 1 Preparation of the mRNA Vaccine

1. Preparation of DNA and mRNA Constructs

-   -   For the present examples a DNA sequence, encoding Gallus gallus        ovalbumin mRNA (R1710) was prepared and used for subsequent in        vitro transcription reactions.    -   According to a first preparation, the DNA sequence coding for        the above mentioned mRNA was prepared. The construct was        prepared by modifying the wild type coding sequence by        introducing a GC-optimized sequence for stabilization, followed        by a stabilizing sequence derived from the alpha-globin-3′-UTR        (muag (mutated alpha-globin-3′-UTR)), a stretch of 64 adenosines        (poly-A-sequence), a stretch of 30 cytosines (poly-C-sequence),        and a histone stem loop. In SEQ ID NO: 2 (see FIG. 3) the        sequence of the corresponding mRNA is shown.

2. In vitro Transcription

-   -   The respective DNA plasmid prepared according to Example 1 was        transcribed in vitro using T7 polymerase. Subsequently the mRNA        was purified using PureMessenger® (CureVac, Tübingen, Germany).

3. Reagents

-   -   Complexation Reagent: protamine

4. Preparation of the Vaccine

-   -   The mRNA R1710 was complexed with protamine by addition of        protamine to the mRNA in the ratio (1:2) (w/w) (adjuvant        component). After incubation for 10 min, the same amount of free        mRNA R1710 used as antigen-providing RNA was added.

OVA-RNActive vaccine (R1710): comprising an adjuvant componentconsisting of mRNA coding for Gallus gallus ovalbumin (R1710) accordingto SEQ ID NO. 2 complexed with protamine in a ratio of 2:1 (w/w) and theantigen-providing free mRNA coding for Gallus gallus ovalbumin (R1710)according to SEQ ID NO. (ratio 1:1; complexed RNA:free RNA).

Example 2 Combination of an Anti-PD1 Antibody and an RNA Vaccine

On day zero, C57BL/6 mice were implanted subcutaneously (right flank)with 3×10⁵ E.G.7-OVA cells per mouse (volume 100 μl in PBS). E.G.7-OVAis a mouse T cell lymphoma cell line stably expressing Gallus gallusovalbumin (OVA). Intradermal vaccination with the RNA vaccine comprisingOVA mRNA R1720 (32 μg/mouse/vaccination day) (according to Example 1) orRinger-lactate (RiLa) as buffer control and treatment with theanti-PD-1/CD279 monoclonal antibody (100 μg i.p.) or an isotype controlaccording to Table 1 started on day 4 and was repeated on days 7, 11,14, 18 and 21. Animals received the antibody injection in the morningand were vaccinated in the afternoon with a minimum of four hoursbetween the treatments.

TABLE 1 Animal groups Number Injected RNA per Injected antibody perGroup of mice vaccination day and mouse treatment day and mouse A 10100% Ringer Lactate — (RiLa) buffer B 10 32 μg — C 6 — 100 μg anti-PD-1D 6 32 μg 100 μg anti-PD-1 E 6 32 μg 100 μg control-IgG2a

The anti-PD-1/CD279 antibody (clone RMP1-14, rat IgG2a) and the isotypecontrol antibody (clone 2A3, rat IgG2a) were purchased from BioXCell(West Lebanon, N.H., USA).

Tumour growth was monitored by measuring the tumour size in 2 dimensions(length and width) using a calliper (starting on day 4). Tumour volumewas calculated according to the following formula:

${{volume}\mspace{14mu} \left( {mm}^{3} \right)} = \frac{{length}\mspace{14mu} ({mm}) \times \pi \times {{width}^{2}\left( {mm}^{2} \right)}}{6}$

The results are shown in FIGS. 1 and 2.

As can be seen in FIG. 1, the OVA mRNA vaccine (OVA-RNActive R1710)alone or in combination with control-IgG delayed tumor growth byapproximately 5 days compared to the buffer-treated control group.Treatment with anti-PD-1 antibody alone was comparable to vaccinationalone, whereas simultaneous application of the RNA vaccine/anti-PD-1combination led to significant inhibition of tumor growth, indicating asynergistic effect. Lines represent the development of mean tumor volumeand error bars the SEM. Statistical analysis was based on the 2way Anovawith Bonferroni posttest.

As can be seen in FIG. 2, the administration of the OVA mRNA vaccine(OVA-RNActive R1710) or anti-PD-1 antibody alone had already asignificant effect on survival, whereas simultaneous application of theRNA vaccine/anti-PD-1 combination led to 50% survival (3 of 6 animals),indicating a synergistic effect.

1. A method of treating a subject having a cancer or a tumor disease, the method comprising administering to the subject an effective amount of: (i) an immunogenic composition comprising at least one mRNA comprising at least one open reading frame (ORF) encoding at least one tumor antigen; and (ii) a PD-1 pathway inhibitor, said PD-1 pathway inhibitor comprising an antagonistic antibody directed against PD-1 or PD-L1.
 2. The method of claim 1, wherein the PD-1 pathway inhibitor comprises an antagonistic antibody directed against PD-1.
 3. The method of claim 1, wherein the PD-1 pathway inhibitor comprises an antagonistic antibody directed against PD-L1.
 4. The method of claim 1, wherein the at least one open reading frame is codon optimized.
 5. The method of claim 1, wherein the at least one open reading frame of the at least one RNA comprises an increased G/C content relative to the open reading frame of a wild type RNA encoding the tumor antigen.
 6. The method of claim 1, wherein the at least one RNA is complexed with a carrier.
 7. The method of claim 6, wherein the carrier is a cationic, polycationic or polymeric carrier.
 8. The method of claim 7, wherein the carrier comprises protamine.
 9. The method of claim 7, wherein the carrier comprises a cationic or polycationic lipid.
 10. The method of claim 1, wherein the PD-1 pathway inhibitor is administered after said immunogenic composition.
 11. The method of claim 1, wherein the immunogenic composition and the PD-1 pathway inhibitor are administered sequentially.
 12. The method of claim 1, wherein the immunogenic composition and the PD-1 pathway inhibitor are administered concurrently.
 13. The method of claim 12, wherein the immunogenic composition and the PD-1 pathway inhibitor are administered in the same composition.
 14. The method of claim 1, wherein the immunogenic composition and the PD-1 pathway inhibitor are administered via different administration routes.
 15. A kit of parts comprising: an immunogenic composition comprising at least one mRNA comprising at least one open reading frame (ORF) encoding at least one tumor antigen; and (ii) a composition comprising a PD-1 pathway inhibitor, said PD-1 pathway inhibitor comprising an antagonistic antibody directed against PD-1 or PD-L1.
 16. A pharmaceutical composition comprising an effective amount of: (i) at least one mRNA comprising at least one open reading frame (ORF) encoding at least one tumor antigen and (ii) a PD-1 pathway inhibitor, said PD-1 pathway inhibitor comprising an antagonistic antibody directed against PD-1 or PD-L1. 